Use of adenovirus and nucleic acids coding therefor

ABSTRACT

The invention relates to the use of a virus, preferably an adenovirus, for producing a medicament. Said virus is replication-deficient in cells which do not contain YB-1 in the core and codes for an oncogene or oncogene product, especially an oncogene protein, which transactivates at least one viral gene, preferably an adenoviral gene, said gene being selected among the group comprising E1B55kDa, E4orf6, E4orf3, and E3ADP.

The present invention relates to the use of adenoviruses as well as tonucleic acids coding therefor and recombinant viral oncoprotein.

A number of therapeutic concepts are currently used in the treatment oftumors. Apart from using surgery, chemotherapy and radiotherapy arepredominant. All these techniques are, however, associated withconsiderable side effects. The use of replication selective oncolyticviruses provides for a new platform for the treatment of tumors. Inconnection therewith a selective intratumor replication of a viral agentis initiated which results in virus replication, lysis of the infectedtumor cell and spreading of the virus to adjacent tumor cells. As thereplication capabilities of the virus is limited to tumor cells, normaltissue is spared from replication and thus from lysis by the virus.

For the time being, several viral systems are subject to clinic trialsaiming at tumor lysis. One example for such an adenovirus is d11520(Onyx-015) which has been successfully used in clinical phases I and II(Khuri, F. et al. Nature Medicine 6, 879-885, 2000). Onyx-015 is anadenovirus having a completely deleted E1B55kDa gene. The completedeletion of the E1B55kDa protein of the adenovirus is based on thediscovery that replication and thus lysis of cells is possible with anadenoviral vector having a p53 deficiency (Kim, D. et al., Proc. Am.Soc. Clin. Oncol. 17, 391a, 1998), whereby normal cells are not harmed.More particularly, the E1B55kDa gene product is involved in theinhibition of p53, the transport of viral mRNA and the switching off theprotein synthesis of the host cell. The inhibition of p53 occurs viaformation of a complex consisting of p53 and the adenoviral codedE1B55kDa protein and/or a complex consisting of E1B55kDa and E4orf6.p53, coded by TP53, is the starting point for a complex regulatorymechanism (Zambetti, G. P. et al., FASEB J. 7, 855-865, 1993), whichresults, among others, in an efficient inhibition of the replication inthe cell of viruses like adenovirus. The gene TP 53 is deleted ormutated in about 50% of all human tumors which results in the absenceof—desired—apoptosis due to chemotherapy or radiation therapy resultingin an usually unsuccessful tumor treatment.

A further concept of tumorlytic adenoviruses is based on the discoverythat if the E1A protein is present in a specific deleted form orcomprises one or several mutations, which do not affect the binding ofRb/E2F and/or p107/E2F and/or p130/E2F, such adenovirus will not inducethe entry of the infected cells into the S phase and will be capable ofreplicating in tumor cells which do not have a functional Rb protein.Additionally, the E1A protein can be deleted at the N-terminus andcomprise one or several mutations in the region of amino acid positions1 to 76 of the E1A proteins, respectively, in order to inhibit thebinding of E1A to p300 and thus to provide for a selective replicationin tumor cells. These approaches are described in an exemplary manner inEuropean patent EP 0 931 830. Examples for such viruses are AdΔ24,d1922-947, E1Ad/01/07 and CB016 (Howe, J. A. et al., Molecular Therapy2, 485-495, 2000; Fueyo, J. et al., Oncogene 19, 2-12, 2000; Heise, C.et al., Nature Medicine 6, 11341139, 2001; Balague, C. et al., J. Virol.75, 7602-7611, 2001). These adenoviral systems for oncolysis known inthe prior art thus comprise distinct deletions in the E1A protein,whereby such deletions had been made under the assumption that afunctional Rb protein and complexes consisting of inactive Rb proteinand E2F, respectively, would block an efficient in vivo replication andin order to provide an adenoviral replication in vivo inRb-negative/mutated cells only. These adenoviral systems according tothe prior art are based on E1A in order to control in vivo replicationusing the early E2 promoter (engl. E2 early promoter) and free E2F(Dyson, N. Genes & Development, 12, 2245-2262, 1998).

A further form of tumorlytic adenoviral systems is based on the use ofselective promoters for specifically expressing the viral oncogene E1Awhich provides for a selective replication in tumor cells (Rodriguez, R.et al., Cancer Res. 57, 2559-2563, 1997).

As described above, the selection of a cellular background which isappropriate for the respective concept underlying the mode of action isimportant for the various concepts of adenoviral tumorlytic viruses. Inother words, the various adenoviral systems currently known may only beused if distinct molecular biological prerequisites are realized. Thislimits the use of such systems to distinct patient groups.

A particular problem in the treatment of tumor diseases arises once thepatients develop a so-called multidrug resistance (engl. multidrugresistance (MDR)) which represents a particularly well studied form ofresistance of tumors against cytostatics (Gottesman and Pastan, Annu.Rev. Biochem. 62, 385-427, 1993). It is based on the overexpression ofthe membrane-bound transport protein P-glycoprotein which belongs to theso-called ABC transporters (Stein, U. et al., JBC 276, 28562-69, 2001,J. Wijnholds, Novartis Found Symp., 243, 69-79, 2002). Bargou, R. C. etal. and Oda, Y. et al (Bargou, R. C. et al., Nature Medicine 3, 447-450,1997; Clin. Cancer Res. 4, 2273-2277, 1998) were able to show thatnuclear localisation of the human transcription factor YB-1 is directlyinvolved in the activation of the expression of the P-glycoprotein.Further studies confirmed that YB-1 is transported into the nucleus byvarious stress conditions such as UV irradiation, administration ofcytostatics (Koike, K. et al., FEBS Lett 17, 390-394, 1997) andhyperthermia (Stein, U. et al., JBC 276, 28562-69, 2001). Furtherstudies confirmed that the nuclear localisation of YB-1 has an impact onone further ABC transporter. This ABC transporter is referred to as MRP(engl. multidrug resistance-related protein) and is involved in theformation of the so-called atypical non-P-glycoprotein dependentmultidrug resistance (Stein, U. et al., JBC 276, 28562-69, 2001).

The problem underlying the present invention is to provide a technicalteaching and in particular a means which allows to treat an organism,more particularly a human organism and a group of patients,respectively, specifically with tumorlytically active agents. It is afurther problem underlying the present invention to provide a meanswhich is suitable to cause tumorlysis in patients having tumor diseaseswhich are resistant to cytostatics, particularly those which have amultidrug resistance.

According to the present invention the problem is solved in a firstaspect by the use of a virus, preferably an adenovirus, for themanufacture of a medicament, whereby the virus is replication deficientin cells which do not have YB-1 in the nucleus, and the virus codes foran oncogene or oncogene product, preferably an oncogene protein, whichtransactivates at least one viral gene in YB-1 nucleus positive cells,preferably an adenoviral gene, whereby the gene is selected from thegroup comprising E1B55kDa, E4orf6, E4orf3 and E3ADP.

In a second aspect, the problem is solved by the use of a virus,preferably an adenovirus, for the replication in cells which have YB-1in the nucleus, whereby the virus is replication deficient in cellswhich do not have YB-1 in the nucleus and the virus codes for anoncogene or oncogene product, in particular oncogene protein, whichtransactivates at least one viral gene, preferably an adenoviral gene,whereby the gene is selected from the group comprising E1B55kDa, E4orf6,E4orf3 and E3ADP.

In an embodiment of the two uses according to the invention, the virus,preferably the adenovirus, replicates in cells which have YB-1 in thenucleus.

In a further embodiment of the two uses according to the invention theviral oncogene protein is E1A and/or the oncogene is the gene coding forE1A and/or the oncogene protein is E1A.

In a preferred embodiment the viral oncogene protein E1A is capable ofbinding to a functional Rb tumor suppressor gene product.

In an alternative embodiment the viral oncogene protein E1A is notcapable of binding to a functional Rb tumor suppressor gene product.

In a further embodiment of the two uses according to the invention theviral oncogene protein E1A is not inducing nuclear localisation of YB-1.

In a still further embodiment of the two uses according to the inventionthe medicament is for patients the cells of whom are either Rb-positiveor Rb-negative.

In a preferred embodiment the cells are those cells which are involvedin the formation of the condition which is to be influenced by themedicament.

In a further embodiment of the two uses according to the invention thecells are Rb-negative and are YB-1 positive in the nucleus, preferablyare YB-1 positive in the nucleus independent from the cell cycle.

In a still further embodiment of the two uses according to the inventionthe medicament is for the treatment of tumors.

In a still further embodiment of the two uses according to the inventionthe cells, particularly the cells forming the tumor or parts thereof,are resistant to drugs, in particular have a multidrug resistance,preferably a resistance against anti-tumor agents and more preferablyagainst cytostatics.

In a preferred embodiment of the two uses according to the invention thecells are expressing, preferably overexpressing the membrane-boundtransport protein P-glycoprotein and/or MRP.

In a further embodiment of the two uses according to the invention thecells are p53-positive or p53-negative.

In an embodiment of the two uses according to the invention the oncogeneprotein has, compared to the wildtype oncogene protein E1A, one orseveral mutations or deletions, whereby the deletion is preferablyselected from the group comprising deletions of the CR3 region anddeletions of the N-terminus and deletions of the C-terminus. Inconnection therewith it is preferred that the E1A oncogene protein canbind to Rb.

In a further embodiment of the two uses according to the invention theoncogene protein has, compared to the wildtype oncogene protein, one orseveral mutations or deletions, whereby the deletion is preferably inthe CR1 region and/or the CR2 region. It is within the invention thatthe oncogene protein E1A is incapable of binding to Rb.

In an embodiment of the two uses according to the invention the viraloncogene protein, in particular E1A, is under the control of atissue-specific and/or tumor-specific promoter.

In a further embodiment of the two uses according to the invention, thevirus, in particular the adenovirus, codes for YB-1.

In a still further embodiment of the two uses according to theinvention, YB-1 is under the control of a tissue-specific and/ortumor-specific promoter.

In a preferred embodiment of the two uses according to the invention,the virus, in particular the adenovirus, codes at least for one proteinwhich is selected from the group comprising E4orf6, E4orf3, E1B55kDa andadenoviral E3ADP protein.

In an alternative embodiment of the two uses according to the invention,the cells have YB-1 in the nucleus, in particular the cells forming thetumor or part thereof have YB-1 in the nucleus.

In a further embodiment of the two uses according to the invention, thetumor has YB-1 in the nucleus upon inducing the transport of YB-1 intothe nucleus.

In a preferred embodiment of the two uses according to the invention,the transport of YB-1 into the nucleus is triggered through at least onemeasure selected from the group comprising radiation, administration ofcytostatics and hyperthermia.

In a particularly preferred embodiment of the two uses according to theinvention, the measure is applied to a cell, an organ or an organism.

In a preferred embodiment of the two uses according to the invention,the virus, in particular the adenovirus, is selected from the groupcomprising AdΔ24, d1922-947, E1Ad/01/07, d11119/1131, CB 016, d1520 andviruses which are lacking an expressed viral E1A oncogene which iscapable of binding a functional Rb tumor suppressor gene product.

In a third aspect the problem is solved by the use of a virus,preferably an adenovirus, for the manufacture of a medicament, wherebythe virus, preferably the adenovirus, is designed such that thereplication is controlled through or by means of YB-1 through theactivation of the E2-late promoter, preferably predominantly through theactivation of the E2-late promoter. In an embodiment YB-1 is either atransgenic YB-1 or a cellular, in particular cellular deregulated YB-1.A transgenic YB-1 is preferably meant to be a YB-1 which is expressed ina cell by a vector, preferably a or the adenovirus. The E2-late promoteris preferably the adenoviral E2-late promoter as present in the wildtypeadenovirus, or an E2-late promoter as described herein in connectionwith the expression of transgenes.

In a fourth aspect the problem is solved by the use of a virus andparticular an adenovirus, for the replication in cells which have YB-1in the nucleus, whereby the virus, in particular the adenovirus, isdesigned such that the replication is controlled by YB-1 through theactivation of the E2-late promoter, preferably predominantly through theactivation of the E2-late promoter. In an embodiment YB-1 is either atransgenic YB-1 or a cellular, in particular cellular deregulated YB-1.A transgenic YB-1 as used herein is preferably a YB-1 which is expressedin a cell by a vector, preferably a or the adenovirus. The E2-latepromoter is preferably the adenoviral E2-late promoter as present in thewildtype adenovirus, or an E2-late promoter as described herein inconnection with the use of the expression of transgenes.

In a preferred embodiment of the third and/or fourth aspect of thepresent invention the adenovirus is designed such as disclosed herein,particularly such as it is designed in order to be used in accordancewith the present invention.

In a fifth aspect the problem is solved by a viral oncogene protein, inparticular an isolated viral oncogene protein which has the followingcharacteristics:

a) transactivation of at least one viral gene in YB-1 nucleus-positivecells, which is selected from the group comprising E1B55kDa, E3ADP andE4orf6 and E4orf3, and

b) lacking induction of YB-1 in the nucleus, in particular the nucleusof the cell in which the viral oncogene protein is present.

In an embodiment the viral oncoprotein is E1A.

In a further embodiment the viral oncogene protein has, compared to thewildtype oncogene protein, one or several mutations or deletions,whereby the deletion is preferably selected from the group comprisingdeletion of the CR3 region, deletion of the N-terminus and deletion ofthe C-terminus.

In an embodiment the induction of YB-1 through the viral oncogeneprotein is absent when E4orf6 and/or E1B55kDa are not present in thenucleus exhibiting cell.

In connection therewith it is intended that the viral oncogene proteinis capable of binding to Rb.

In an alternative embodiment the viral oncogene protein comprises one orseveral mutations or deletions, whereby the deletion is preferably inthe CR1 region and/or the CR2 region of the E1A oncogene protein. Inconnection therewith it is intended that the viral oncogene protein isnot able to bind to Rb.

In a sixth aspect the invention is related to the use of a viralreplication system, preferably an adenoviral replication system,comprising a nucleic acid which codes for a virus, in particular anadenovirus as used in accordance with the present invention, andcomprising one nucleic acid of a helper virus, whereby the nucleic acidof the helper virus comprises a nucleic acid which codes for YB-1.

In an embodiment the viral nucleic acid, in particular the adenoviralnucleic acid, and/or the nucleic acid of the helper virus are present asa vector which can replicate.

In a seventh aspect the invention is related to the use of a nucleicacid coding for a virus, in particular an adenovirus, as it is used inaccordance with the invention, for the manufacture of a medicament, inparticular for the manufacture of a medicament for the treatment oftumors.

In an embodiment the cells, in particular the cells forming the tumor orparts thereof, are resistant, in particular have a multidrug resistance,against drugs, preferably anti-tumor agents, and more preferablycytostatics.

In an eighth aspect the invention is related to the use of a nucleicacid which codes for a virus, in particular an adenovirus, as is used inaccordance with the present invention, for the replication in cellswhich have YB-1 in the nucleus, whereby the virus is replicationdeficient in cells which do not have YB-1 in the nucleus, and the viruscodes for an oncogene or oncogene product which transactivates at leastone viral gene, preferably an adenoviral gene, in YB-1 nucleus-positivecells, whereby the gene is selected from the group comprising E1B55kDa,E4orf6, E4orf3 and E3ADP.

In a ninth aspect the problem is solved by the use of a nucleic acidwhich codes for a virus, preferably an adenovirus, as is used inaccordance with the invention, for the manufacture of a medicament,whereby the virus is designed such that the replication is controlled byYB-1 through the activation of the E2-late promoters, preferablypredominantly through the activation of the E2-late promoter. In anembodiment the YB-1 is either a transgenic YB-1 or a cellular, inparticular cellular deregulated YB-1. A transgenic YB-1 as used hereinis preferably a YB-1 which is expressed in a cell by a vector,preferably a or the adenovirus. The E2-late promoter is preferably theadenoviral E2-late promoter as is present in the wildtype adenovirus, oran E2-late promoter as described herein in connection with the use ofthe expression of transgenes.

In a tenth aspect the problem is solved by the use of a nucleic acidwhich codes for a virus, in particular an adenovirus, as used inaccordance with the invention for replication in cells, whereby thevirus is designed such that the replication is controlled by YB-1through the activation of the E2-late promoter, preferably predominantlythrough the activation of the E2-late promoter. In an embodiment theYB-1 is either a transgenic YB-1 or a cellular, in particular cellularderegulated YB-1. As used herein, transgenic YB-1 is preferably a YB-1which is expressed by a vector in a cell, preferably by a or theadenovirus. The E2-late promoter is preferably the adenoviral E2-latepromoter as present in wildtype adenovirus, or an E2-late promoter asused in connection with the expression of transgenes described herein.

In an eleventh aspect the problem is solved by the use of a vectorcomprising one of the previously described nucleic acids, for the use inaccordance with the first or second aspect of the present invention.

In a twelfth aspect the invention is related to the use of an agentinteracting with YB-1 for the characterisation of cells, cells of atumor tissue or patients, in order to determine whether these shall becontacted and/or treated with a virus, in particular an adenovirus,which is used in accordance with the invention.

In an embodiment the agent is selected from the group comprisingantibodies, anticalines, aptamers, aptazymes and spiegelmers.

In a thirteenth aspect the problem is solved by the use of the viraloncogene protein according to the present invention or a nucleic acidcoding therefor, for the manufacture of a virus, in particular anadenovirus, which is used in accordance with the first and second aspectof the present invention.

In an embodiment the virus comprises a nucleic acid coding for atransgene.

In a further embodiment the virus comprises the translation productand/or the transcription product of a transgene.

In a preferred embodiment the nucleic acid of the adenoviral replicationsystem and/or the nucleic acid of the helper virus comprises a transgeneor a nucleic acid coding for a transgene.

In a still further embodiment the nucleic acid comprises a transgene ora nucleic acid coding for a transgene.

In an alternative embodiment the transgene is selected from the groupcomprising prodrug genes, cytokines, apoptosis-inducing genes, tumorsuppressor genes, genes for metalloproteinases inhibitors and genes forangiogenesis inhibitors.

In an embodiment the transgene is selected from the group comprisingnucleic acids for siRNA, for aptamers, for antisense molecules and forribozymes, whereby the siRNA, the aptamers, the antisense moleculesand/or the ribozymes are targeted against a target molecule.

In a further embodiment the target molecule is selected from the groupcomprising resistance relevant factors, anti-apoptosis factors,oncogenes, angiogenesis factors, DNA synthesis enzymes, DNA repairenzymes, growth factors and their receptors, transcription factors,metalloproteinases, in particular matrix metalloproteinases, andplasminogen activator of the urokinase type. In an embodiment theresistance-relevant factors are preferably selected from the groupcomprising P-glycoprotein, MRP and GST, and also comprise nucleic acidscoding therefor. In an embodiment the anti-apoptosis factors areselected from the group comprising BCL2, and also comprise the nucleicacids coding therefor. In an embodiment the oncogenes are selected fromthe group comprising Ras, in particular mutated Ras, Rb and Myc, andalso comprise nucleic acids coding therefor. In an embodiment theangiogenesis factors are selected from the group comprising VEGF and HMGproteins and also comprise the nucleic acids coding therefor. In anembodiment the DNA synthesis enzymes are selected from the groupcomprising telomerase and also comprise nucleic acids coding therefor.In an embodiment the DNA repair enzymes are selected from the groupwhich comprises Ku-80, and also comprise nucleic acids coding therefor.In an embodiment the growth factors are selected from the groupcomprising PDGF, EGF and M-CSF, and comprise also nucleic acids codingtherefor. In an embodiment the receptors are in particular receptors forgrowth factors, whereby the growth factors are preferably selected fromthe group comprising PDGF, EGF and M-CSF, and also comprise the nucleicacids coding therefor. In an embodiment the transcription factors areselected from the group comprising YB-1, and also comprise the nucleicacid coding therefor. In an embodiment the metalloproteinases arepreferably matrix metalloproteinases. In a preferred embodiment thematrix metalloproteinases are selected from the group comprising MMP-1and MMP-2, and also comprise the nucleic acids coding therefor. In anembodiment the plasminogen activators of the urokinase type are selectedfrom the group comprising uPa-R, and also comprise the nucleic acidscoding therefor.

In a still further embodiment the medicament comprises additionally atleast one pharmaceutically active compound.

In a preferred embodiment the pharmaceutically active compound isselected from the group comprising cytokines, metalloproteinaseinhibitors, angiogenesis inhibitors, cytostatics and cell cycleinhibitors.

The present invention is based on the surprising finding that the DNAreplication of E1A-modified adenoviruses in YB-1 nucleus-positive tumorcells is based on the activation of the E2-late promoter. E1A-modifiedadenoviruses as used herein, are adenoviruses which (a) do not replicatein YB-1 nucleus-negative cells or show a reduced, preferably a stronglyreduced replication in YB-1 nucleus-negative cells compared to therespective wildtype, (b) transactivate at least one viral gene, wherebythe gene is in particular selected from the group comprising E1B55,E4orf6, E4orf3 and E3ADP, and/or (c) do not translocate cellular YB-1through the adenovirus into the nucleus. Optionally the adenovirusesused in accordance with the present invention have the furthercharacteristic that the binding of the adenoviral encoded E1A proteininterferes with the binding of E2F to Rb and is able to dissolve therespective complex consisting of E2F and Rb, respectively. Adenoviruseswhich have at least one or several of the aforementioned features a) toc), preferably all of features a) to c), are replication deficient incells which do not have YB-1 in the nucleus.

In an embodiment a strongly reduced replication as used hereinparticularly means a replication which, compared to the wildtype, isreduced by a factor of 2, preferably by a factor of 5, more preferablyby a factor of 10 and most preferably by a factor of 100. In a preferredembodiment such comparison of the replication is performed usingidentical or similar cell lines, identical or similar virus titers forinfection (multiplicity of infection, MOI, or plaque forming unit, pfu)and/or identical or similar general experimental conditions. Replicationas used herein particularly means formation of particles. In a furtherembodiment the measure for replication can be the extent of viralnucleic acid synthesis. Methods for the determination of the extent ofthe viral nucleic acid synthesis as well as methods for determiningparticle formation are known to the ones skilled in the art.

The findings, methods, uses or nucleic acids, proteins, replicationsystems and the like described herein are not necessarily limited toadenoviruses. In principle, such systems exist also in other viruseswhich are herewith also comprised.

A replication which is comparable to wildtype replication, can berealized upon an infection rate of 1 to 10 pfu/cell compared to 10 to100 pfu/cell according to the prior art when using the viruses accordingto the present invention or when using the viruses described herein inaccordance with the present invention.

Cellular YB-1 as used herein shall mean any YB-1 which is coded by acell and preferably is also expressed by a cell, whereby this YB-1 ispresent in the cell, preferably prior to the infection of the respectivecell with an adenovirus, preferably an adenovirus and/or a helpervirusas described herein. It is, however, also within the present inventionthat cellular YB-1 is a YB-1 which is introduced into the cell orproduced by such cell upon application of exogenous measures such as, e.g., infection with a virus, in particular with an adenovirus.

Without wishing to be bound by this in the following, the presentinventor assumes that the E2-early promoter, i. e. the early E2 promoteris not switched on through the human cellular E2F transcription factorin connection with the replication of the viruses used herein inaccordance with the present invention. The switching on of thereplication is independent of the Rb status of the cells, i. e. whichmeans that the tumor cells which are infected using the virusesdisclosed herein and which are preferably lysed subsequently thereafter,may comprise both functional as well as inactive Rb proteins.Additionally, adenoviral replication does neither need any functionalp53 protein nor is it affected by its presence, when using theadenoviruses disclosed herein or under the conditions disclosed herein.Insofar, the technical teaching departs from the principle underlyingthe use of the oncolytic or tumorlytic adenoviruses of the AdΔ24,d1922-947, E1Ad/01/07, CB016 type or of those adenoviruses which are,for example, described in European patent EP 0 931 830, and into whichone or several deletions have been introduced into the E1A protein underthe assumption that intact functional Rb proteins are an obstacle to anefficient replication in vivo thus providing an adenoviral replicationin vivo only in Rb-negative and Rb-mutated cells, respectively. Theseadenoviral systems according to the prior art are based on E1A in orderto control in vivo replication of adenoviruses by means of the early E2promoter (E2 early promoter) and “free E2F”. Nevertheless, these virusesaccording to the prior art may be used in accordance with the presentinvention, i. e. for replication in cells which contain YB-1 in thenucleus independent from the cell cycle.

The viruses described in said European patent EP 0 931 830 and inparticular adenoviruses may be used in accordance with the presentinvention. More particularly, the viruses described in said patent arereplication deficient and lack an expressed viral oncoprotein which iscapable of binding a functional Rb tumor suppressor gene product. Theadenovirus can particularly be an adenovirus which is lacking expressedviral E1A oncoprotein which is capable of binding a functional tumorsuppressor gene product, in particular Rb. The viral E1A oncoprotein cancomprise an inactivating mutation, for example in the CR1 domain atamino acid positions 30 to 85 in Ad 5, nucleotide positions 697 to 790and/or the CR2 domain at amino acid positions 120 to 139 in Ad 5,nucleotide positions 920 to 967 which are involved in the binding ofp105 Rb protein, p130 and p107 protein. It can also be intended that theadenovirus is of type 2 d1 312 or the adenovirus is of type 5 NT d11010.

Replication ultimately occurs in cells which comprise YB-1 in thenucleus, preferably independent from the cell cycle, which are thus YB-1nucleus-positive, when using adenoviruses in accordance with theinvention for the manufacture of a medicament, in particular for themanufacture of a medicament for the treatment of tumor diseases, andwhen using adenoviruses in accordance with the invention for replicationin cells which have YB-1 in the nucleus. It is particularly noteworthythat the adenoviruses as such do not replicate in cells which do nothave YB-1 in the nucleus but have YB-1 essentially in the cytoplasmonly, or replicate at a significantly reduced level. Insofar it isnecessary that YB-1 is present in the nucleus for a successfulreplication of these viruses. This can, for example, as will be outlinedin the following in more detail, be realized by applying measures to thecells which result in the expression of YB-1 in the nucleus or in thepresence of YB-1 in the nucleus. A respective measure can, for example,be the coding and expression, respectively, of YB-1 through adenovirusesused in accordance with the present invention which in addition to theadenoviral genes also comprise a genetic information coding for YB-1 andin particular for the expression of YB-1. Other measures which result intransport, induction or expression of YB-1 in the nucleus of the cell,are stress conditions such as administration of cytostatics,irradiation, hyperthermia and the like, to the cell and to an organismcontaining such cell.

The adenoviruses which are used in connection with the presentinvention, in particular for tumor lysis, are further characterized suchthat they do not replicate in cells which do not have YB-1 in thenucleus, in other words which are YB-1 nucleus-negative.

A further feature of the adenoviruses which are to be used in accordancewith the invention, is that they code for a viral oncoprotein which isalso referred to herein as oncogene protein, whereby the oncogeneprotein is preferably E1A, whereby the oncogene protein, is capable ofactivating at least one viral gene which can have an impact on thereplication of the virus and/or cell lysis of the cells infected by thevirus. It is preferred that the influence on replication is such thatthe virus replicates better in the presence of the oncogene proteincompared to a situation where the oncogene protein of the respectivevirus is lacking. This process is referred to herein also astransactivating and in particular E1A transactivating, when thetransactivation is mediated through E1A. The term “transactivate” or“transactivation” describes preferably the process that the respectiveviral oncoprotein has an impact on the expression and/or thetranscription of one or several other genes different from the viraloncoprotein coding gene itself, i. e. is preferably controlling itsexpression and/or translation, and in particular activates this/these.Such viral genes are preferably E1B55kDa, E4orf6, E4orf3 and E3ADP aswell as any combination of the aforementioned genes and gene products,respectively.

A further, although preferably optional, feature of the adenoviruses tobe used in accordance with the invention, is the binding to and of tumorsuppressor Rb. In principle it is within the present invention that theadenoviruses used in accordance with the present invention bind to Rb ordo not bind to Rb. The use of both alternative embodiments of theadenoviruses is possible independently from the Rb status of the cell tobe treated.

In order to confer the capability to not bind to Rb, the followingdeletions of the E1A oncoprotein are, for example, possible: Deletion inthe CR1 region (amino acid positions 30-85 in Ad5) and deletion of theCR2 region (amino acid positions 120-139 in AD5). In doing so, the CR3region is maintained and can have its transactivating function on theother early viral genes.

In contrast thereto, the following deletions to the E1A oncoprotein arein principle possible in order to impart E1A the capability to bind toRb: deletion of the CR3 region (amino acid positions 140-185); deletionof the N-terminus (amino acid positions 1-29); deletion of amino acidpositions 85-119; and deletion of the C-terminus (amino acid positions186-289). The regions recited herein do not interfere with the bindingof E2F to Rb. The transactivating function remains, however, is reducedcompared to wildtype Ad5.

Such viruses which are known in the prior art are generally regarded asreplication deficient. It is, however, the merit of the present inventorthat he has recognised that they are capable of replication in asuitable background nevertheless, in particular a cellular background.Such a suitable cellular background is caused or provided by thepresence of YB-1 in the nucleus, preferably a cell cycle independentpresence of YB-1 in the nucleus. The term cells or cellular systems, asused herein, comprises fragments of cells or fractions of cell lysatesas well as cells which are present in vitro, in vivo or in situ.Insofar, the term cellular systems or cells also comprises cells whichare present in a cell culture, tissue culture, organ culture or in anyother tissue or organ in vivo and in situ, respectively, isolated, ingroups or as part of tissues, organs or organisms or are also present assuch in a preferably living organism. The organism is preferably avertebrate organism and more preferably a mammal. It is particularlypreferred that the organism is a human organism.

Additionally, it is within the present invention that based on thetechnical teaching provided herein, new viruses are generated which havethe replication characteristic of the adenoviruses described herein aswell as the one of adenoviruses of the prior art in cells which are YB-1nucleus-positive. In other words, preferably starting from theadenoviruses already known further viruses can be designed which havethe features defined herein needed for their use in accordance with thepresent invention.

In connection with the present invention the modified E1A oncoprotein ofthe various adenoviruses which are to be used in accordance with theinvention, is capable of transactivating the early viral genes such as,for example, E1B55kDa, E4orf3, E4orf6, E3ADP, in YB-1 nucleus-positivecells. In connection therewith, there are preferably otherwise nofurther changes to the viral genome and the respective adenovirus canotherwise correspond to an adenovirus of the wildtype or any derivativethereof.

The viruses disclosed herein which code for a transactivating oncogeneprotein in the sense of the present invention or which comprise suchoncogene protein, comprise, for example, the adenoviruses AdΔ24,d1922-947, E1Ad/01/07, CB106 and/or the adenoviruses described inEuropean patent EP 0 931 380, which are each capable of transactivatingthe early genes, such as E1B, E2, E3 and/or E4, and are comparable toadenoviruses of the wildtype, in particular wildtype Ad5. A particularregion of the E1A protein is responsible for transactivation in thesecases. Within various adenovirus serotypes there are three highlyconserved regions in the E1A protein. The CR1 region from amino acidpositions 41-80, the CR2 region from amino acid positions 120-139 andthe CR3 region from of amino acid positions 140-188. The transactivatingfunction is primarily based on the presence of the CR3 region in the E1Aprotein. The amino acid sequence of CR3 is unaltered in theaforementioned adenoviruses. This results in a transactivation of theearly genes E1B, E2, E3 and E4 independent from the presence of YB-1 inthe nucleus or in the cytoplasma.

In the recombinant adenovirus d1520, however, the CR3 region has beendeleted. Thus d1520 expresses a so-called E1A12S protein which does notcomprise the amino acid sequence of the CR3 region. As a consequence,d1520 can exert a very weak transactivating function only, in particularon the E2 region, and thus does not replicate in YB-1 nucleus-negativecells. In YB-1 nucleus-positive cells YB-1 is transactivating the E2region and thus allows an efficient replication of d1520. This is thebasis for the use of systems like d1520 and of systems on the basis ofd1520 for the purposes disclosed herein, respectively. A furtherimportant difference between both the previously described groups ofadenoviruses, i. e. delta 24 (herein also referred to as AdΔ24) andd1520 resides in the fact that with d1520 the early genes E1B, E3 and E4are more strongly transactivated in YB-1 nucleus-positive cells comparedto YB-1 nucleus-negative cells. In contrast, there are no or only minordifferences with delta 24. The transactivation effect of d1520 and moreparticularly of the E1A12S protein, however, is significantly reducedcompared to wildtype adenovirus. This transactivation is, however,sufficient in order to allow for an efficient replication in YB-1nucleus-positive cells, as shown in example 10. The design of the E1Aprotein and of the nucleic acid coding therefor described herein and inparticular in this context such that the E1A protein has one or severaldeletions and/or mutations compared to the wildtype oncogene proteinE1A, whereby the deletion is preferably one selected from the groupcomprising deletions of the CR3 region and deletions of the N-terminusand deletions of the C-terminus, including and particularly preferredthose embodiments of the E1A protein as described in connection withd1520 or AdΔ24, d1922-947, E1Ad/01/07, CB106 and/or the adenovirusesdescribed in European patent EP 0 931 830, are embodiments of viruses,in particular adenoviruses, the replication of which is controlled byYB-1 through the activation of the E2-late promoter, preferablypredominantly through the activation of the E2-late promoter. Furtherembodiments of the E1A protein which allow this form of replication ofadenoviruses, can be generated by the ones skilled in the art based onthe disclosure provided herein.

In further adenoviruses which are to be newly constructed, which arealso referred to herein as derivatives and which may be used inaccordance with the present invention, typically have an E1 deletion, anE1/E3 deletion and/or an E4 deletion, i. e. the correspondingadenoviruses are not able to generate functionally active E1 and/or E3and/or E4 expression products and respective products, respectively, or,in other words, these adenoviruses are only capable to generatefunctional inactive E1, E3 and/or E4 expression products, whereby afunctionally inactive E1, E3 and/or 4 expression product as such whichis either not present as an expression product at all, whether at thetranscription level and/or the translation level, or it is present in aform in which it at least is lacking one of the functions it has inwildtype adenoviruses. The function(s) of the expression product of thewildtype adenovirus is/are known to the ones skilled in the art and, forexample, described in Russell, W. C., Journal of Virology, 81,2573-2604, 2000. Russell (supra) describes also principles for theconstruction of adenoviruses and adenoviral vectors which areincorporated herein by reference. It is also within the presentinvention that the modified E1A oncoprotein, E1B55kDa, E4orf6 and/orE3ADP (adenoviral death protein (ADP)) (Tollefson, A. et al., JVirology, 70, 2296-2306, 1996) is expressed in such a vector eitherindividually or in any combination. In connection therewith, theindividually named genes as well as the transgenes disclosed herein, canbe cloned into the E1 and/or E3 and/or E4 region and be expressedindependently by virtue of a suitable promoter or under the control of asuitable promoter. Basically, the regions E1, E3 and E4 are similarlysuitable as cloning sires within the adenoviral nucleic acid. Suitablepromoters are, among others, those as disclosed herein in connectionwith the control and expression, respectively, of E1A, in particular ofthe modified E1A.

Finally, in one embodiment the adenoviruses which are to be used inaccordance with the present invention, are deficient with regard to E1B,in particular with regard to E1B 19 kDa.

As used herein, the term deficient generally means a condition in whichE1B does not have all of the characteristics inherent to the wildtypebut at least one of these characteristics is absent.

The adenoviruses which are used in accordance with the inventiondisclosed herein, are, basically, known in the prior art in someembodiments. The adenoviruses used in accordance with the presentinvention are preferably recombinant adenoviruses, particularly alsowhen a change, compared to the wildtype, has been made in accordancewith the technical teaching provided herein. It is within the skills ofthose of the art to delete or mutate those adenoviral nucleic acidsequences which are not essential for the present invention. Suchdeletions may, for example, be related to a part of the nucleic acidcoding for E3 and E4 as also described herein. A deletion of E4 isparticularly preferred if such deletion does not extend to the proteinE4orf6, or, in other words, the adenovirus to be used in accordance withthe present invention codes for E4orf6. In preferred embodiments theseadenoviral nucleic acids may still be packed into the viral capsid andmay thus form infectious particles. The same is true for the use of thenucleic acids in accordance with the present invention. It should benoted that in general the adenoviral systems may be deficient withregard to single or several expression products. In connection therewithit is to be taken into consideration that this may be either based onthe fact that the nucleic acid coding for such expression product iscompletely mutated or deleted or mutated or deleted to the extent thatessentially no expression product is produced anymore or based on thelack of promoters or transcription factors which control the expression,or which are active in a manner different from wildtype, either at thenucleic acid level (lack of a promoter; cis-acting element) or at thetranslation system and the transcription system, respectively(trans-acting elements). Particularly the latter aspect may be dependenton the cellular background.

Apart from using adenoviruses in accordance with the present invention,which are already known, also novel adenoviruses can be used to the sameextent as has already been disclosed for the other adenovirusesdescribed herein. The novel adenoviruses according to the inventionresult from the technical teaching provided herein. Particularlypreferred representatives are, for example, the viruses Xvir03 andXvir03/01 depicted in FIG. 16 and FIG. 17, the design principle of whichis also further illustrated in examples 11 and 12.

In the case of vector Xvir03 a CMV promoter is cloned into the E1 regionwhich codes the nucleic acids for E1B55kDa and E4orf6, which areseparated by a IRES sequence. Due to the introduction of these two genesand the gene products produced therefrom, respectively, a replicationefficiency is created which factually corresponds to the one of wildtypeviruses, whereby the selectivity of the replication is maintained forcells, particularly tumor cells, insofar as a replication happens inparticular in YB-1 nucleus-positive cells and more particularly in cellsin which YB-1 is deregulated. Cells in which YB-1 is deregulated, arepreferably those which show an increased expression of YB-1, preferablycompartment-independent, compared to normal or non-tumor cells.

A further development of virus Xvir03 is virus Xvir03/01 into which, ina preferred embodiment, therapeutic genes or transgenes are cloned underthe control of a specific promoter, in particular a tumor-specific ortissue-specific promoter. It is also within the scope of such a virusthat also the E4 region is functionally inactive, preferably is deleted.The transgenes described herein may also be cloned into the E4 region,whereby this may occur in addition or alternative to the cloning of atransgene into the E3 region.

Such therapeutic genes may be prodrug genes, genes for cytokines,apoptosis-inducing genes, tumor suppressor genes, genes formetalloproteinase inhibitors and/or angiogenesis inhibitors.Additionally, siRNA, aptamers, antisense and ribozymes may be expressedwhich are directed against cancer-relevant target molecules. Preferably,the single or the multiple target molecules is/are selected from thegroup comprising resistance relevant factors, anti-apoptosis factors,oncogenes, angiogenesis factors, DNA synthesis enzymes, DNA repairenzymes, growth factors and their receptors, transcription factors,metalloproteinases, in particular matrix metalloproteinases andplasminogen activator of the urokinase type. Preferred embodimentsthereof have already been disclosed herein.

Possible prodrug genes, which may be used in preferred embodiments, are,for example, cytosine deaminase, thymidine kinase, carboxypeptidase,uracil phosphoribosyl transferase; purine nucleoside phosphorylase(PNP); Kim et al, Trends in Molecular Medicine, Volume 8, No. 4 (Suppl),2002; Wybranietz W. A. et al., Gene Therapy, 8, 1.654-1664, 2001;Niculescu-Duvaz et al., Curr. Opin. Mol. Therapy, 1, 480.486, 1999;Koyama et al., Cancer Gene Therapy, 7, 1015-1022, 2000; Rogers et al.,Human Gene Therapy, 7, 2235-2245, 1996; Lockett et al., Clinical CancerRes., 3, 2075-2080, 1997; Vijayakrishna et al., J. Pharmacol. And Exp.Therapeutics, 304, 1280-1284, 2003.

Possible cytokines which may be used in preferred embodiments, are, forexample, GM-CSF, TNF-alpha, Il-12, Il-2, Il-6, CSF, interferon-gamma;Gene Therapy, Advances in Pharmacology, Volume 40, Editor: J. ThomasAugust, Academic Press; Zhang und Degroot, Endocrinology, 144,1393-1398, 2003; Descamps et al., J. Mol. Med., 74, 183-189, 1996;Majumdar et al., Cancer Gene Therapy, 7, 1086-1099, 2000.

Possible apoptosis inducing genes as may be used in preferredembodiments, are, for example, decorin: Tralhao et al., FASEB 3, 17,464-466, 2003; retinoblastoma 94: Zhang et al., Cancer Res., 63,760-765, 2003; Bax and Bad: Zhang et al, Hum. Gene Ther., 20, 2051-2064,2002; apoptin: Noteborn and Pietersen, Adv. Exp. Med. Biol., 465,153-161, 2000); ADP: Toth et al., Cancer Gene Therapy, 10, 193-200,2003; bcl-xs: Sumantran et al., Cancer Res, 55, 2507-2512, 1995; E4orf4:Braithwaite and Russell, Apoptosis, 6, 359-370, 2001; FasL, Apo-1 andTrail: Boehringer Manheim, Guide to Apoptotic Pathways, Arai et al.,PNAC, 94, 13862-13867, 1997; Bims; Yamaguchi et al., Gene Therapy, 10,375-385, 2003; GNR163: Oncology News, 17 Juni, 2000.

Possible tumor suppressor genes as may be used in preferred embodiments,are, for example, E1A, p53, p16, p21, p27, MDA-7. Opalka et al., CellTissues Organs, 172, 126-132, 2002, Ji et al., Cancer Res., 59,3333-3339, 1999, Su et al., Oncogene, 22, 1164-1180, 2003.

Possible angiogenesis inhibitors as may be used in preferred embodimentsare, for example, endostatin, angiostatin: Hajitou et al., FASEB J., 16,1802-1804, 2002, and antibodies against VEGF (Ferrara, N., Semin Oncol2002 December; 29 (6 Suppl 16): 10-4.

Possible metalloproteinase inhibitors as may be used in preferredembodiments are, for example, Timp-3, Ahonen et al., Mol Therapy, 5,705-715, 2002; PAI-I; Soff et al., J. Clin. Invest., 96, 2593-2600,1995; Timp-1, Brandt K. Curr. Gene Therapy, 2, 255-271, 2002.

siRNA (short interfering RNA) consists of two, preferably two separateRNA strands, which hybridise with each other due to basecomplementarity, i. e. are essentially base paired and preferably have alength of up to 50 nucleotides, preferably between 18 and 30nucleotides, more preferably less than 25 nucleotides and mostpreferably 21, 22 or 23 nucleotides, whereby these figures refer to thesingle strand of the siRNA, in particular to the length of the stretchof the single strand which hybridises with one, and more particularlywith the second single strand and is base paired therewith,respectively. siRNA specifically induces or mediates the degradation ofmRNA. The specificity required thereto for is provided by the sequenceof the siRNA and thus its binding site. The target sequence to bedegraded is essentially complementary to the first or to the second oneof the siRNA forming strands. Although the exact mode of action is stillunclear, it is assumed that siRNA represents a biological strategy forcells to inhibit certain alleles during development and to protectitself from viruses. siRNA mediated RNA interference is used for thespecific suppression or even complete knock-out of the expression of aprotein by introducing a gene specific double-stranded RNA. For higherorganisms an siRNA having a length from 19 to 23 nucleotides, is thusparticularly preferred as it does not result in activation of aninspecific defense reaction, the so-called interleukin response.Immediate transfection of double-stranded RNA consisting of 21nucleotides having symmetric 2-nt long overhangs at the 3′ end was ableto mediate RNA interference in mammal cells and was highly efficientcompared to other technologies such as ribozymes and antisense molecules(Elbashir, S. Harborth J. Lendeckel W. Yalvcin, A. Weber K Tuschl T:Duplexes of 21-nucleotide RNAs mediate RNA interference in culturedmammalian cells. Nature 2001, 411: 494-498). Only very few siRNAmolecules were necessary so as to inhibit the expression of the targetgene. In order to avoid the limitations of exogenously administeredsiRNA which particularly resides in the transient nature of theinterference phenomenon and the specific delivery of the siRNAmolecules, the prior art uses vectors which allow for an endogenoussiRNA expression. For example, oligonucleotides having a length of 64nucleotides are provided which contain the 19 nucleotide long targetsequence, both in sense as well as in antisense orientation, separatedthrough a, for example, 9 nucleotide long spacer sequence which wasintroduced into the vector. The resulting transcript folded into ahairpin structure having a stem structure of, for example, 19 basepairs. The loop is rapidly degraded in the cell so that a functionalsiRNA is generated (Brummelkamp et al., Science, 296, 550-553, 2002).

The activity of pRb and E2F, respectively, is regulated throughphosphorylation. The hypophosphorylated form of pRb is predominantlypresent in the G1 and M phase. In contrast, the hyperphosphorylated formof pRb is present in the S and G2 phase. E2F is released from thecomplex consisting of E2F and hypophosphorylated pRb by phosphorylationof pRb. The release of E2F from the complex consisting of E2F andhypophosphorylated pRb results in the transcription of E2F dependentgenes. The E1A protein does not only bind to the hypophosphorylated formof pRb, whereby the binding of E1A to pRb happens mostly through the CR2region of the E1A protein. Additionally, it also binds to the CR1region, although with lower affinity (Ben-Israel and Kleiberger,Frontiers in Bioscience, 7, 1369-1395, 2002; Helt and Galloway,Carcinogenesis, 24, 159-169, 2003).

The nucleic acid coding for YB-1 which, in an embodiment of theadenoviruses to be used in accordance with the present invention, ispart of the adenoviruses, may also comprise a nucleic acid sequencemediating the transport of YB-1 into the nucleus. The nucleic acids,adenoviruses and adenoviral systems in accordance with the invention aswell as the adenoviruses known in the prior art such as, for example,Onyx-015, AdΔ24, d1922-947, E1Ad/01/07, CB016, d1 520 and theadenoviruses described in patent EP 0 931 830, can be used as such or incombination with these nucleic acids in accordance with the invention inconnection therewith as adenoviruses and adenoviral systems and thus asthe corresponding nucleic acids. Suitable nucleic acid sequences whichmediate nucleus transport, are known to the ones skilled in the art and,for example, described in (Whittaker, G. R. et al., Virology, 246, 1-23,1998; Friedberg, E. C., TIBS 17, 347, 1992; Sans, D. A. et al.,Bioessays 2000 June; 22(6): 532-44; Yoneda, Y., J. Biochem. (Tokyo) 1997May; 121(5): 811-7; Boulikas, T., Crit. Rev. Eukaryot. Gene Expr. 1993;3(3): 193-227; Lyons R H, Mol. Cell Biol., 7, 2451-2456, 1987). Inconnection with the nucleus transport mediating nucleic acid sequences,different principles can be used. One such principle may, for example,be that YB-1 is formed as a fusion protein together with a signalpeptide and is introduced into the nucleus and that the replication ofthe adenoviruses according to the present invention thus occurs.

A further principle which may be realised in the design of theadenoviruses used in accordance with the invention, is that YB-1 can beprovided with a transporter sequence which, preferably starting fromsynthesis in the cytoplasma, introduces YB-1 into the cell nucleus orwhich translocates YB-1 into the cell nucleus, and promotes viralreplication there. An example for a particularly effective nucleic acidsequence mediating nucleus transport is the TAT sequence of HIV whichis, among other suitable nucleic acid sequences of that type describedin Efthymiadis, A., Briggs, L J, Jans, D A., JBC 273, 1623-1628, 1998.It is within the present invention that the adenoviruses which are usedin accordance with the present invention, comprise nucleic acidsequences which code for peptides coding for nuclear transportation.

It is within the present invention that YB-1 is present in its fulllength, particularly in a form which corresponds to the wildtype ofYB-1. It is within the present invention that YB-1 is used or present asa derivative, such as, e. g. in shortened or truncated form. A YB-1derivative as used or present within the present invention, is a YB-1which is capable of binding to the E2-late promoter and thus activatesgene expression of the adenoviral E2 region. Such derivativesparticularly comprise the YB-1 derivatives disclosed herein. Furtherderivatives may be generated by deletion of single or several aminoacids at the N-terminus, at the C-terminus or within the amino acidsequence.

With regard to the previously mentioned various further expressed genesand gene products coded by the adenoviruses, it is also possible thatthese are coded in any combination and expressed in any combination,respectively.

The terms adenovirus and adenoviral system shall have essentially thesame meaning within the present invention. Adenovirus shall particularlyrefer to the complete viral particle comprising the capsid and thenucleic acid. The term adenoviral system particularly focuses on thefact that the nucleic acid shall be changed compared to the wildtype.Preferably such changes comprise changes in the structure of the genomeof the adenovirus as may arise from deleting and/or adding and/ormutating promoters, regulatory sequences and/or coding sequences such asopen reading frames. Additionally, the term adenoviral system ispreferably used in connection with a vector, which is, for example, usedin gene therapy.

The previously provided comments, including any use as well as design ofthe adenoviruses and adenoviral systems, respectively, apply also to thecoding nucleic acids and vice versa.

In connection with the present invention it is possible that theadenoviruses to be used in accordance with the present invention and thenucleic acids coding therefor, respectively, may be any respectiveadenoviral nucleic acid which results in a replication event as such orin combination with further nucleic acid sequences. It is possible, asexplained herein, that by means of helper virus the sequences and/orgene products required for replication are provided. To the extent it isreferred to coding nucleic acid sequences and to the extent that suchnucleic acid sequences are known, it is within the invention that notonly the identical sequences used but also sequences derived therefrom.The term derived sequences shall in particular refer herein to sequenceswhich still result in a gene product, either a nucleic acid or apolypeptide, that exhibits a function which corresponds to one or thefunction of a non-derived sequence. This can be determined by simpleroutine tests known to the one skilled in the art. An example for suchderived nucleic acid sequences are those nucleic acid sequences whichcode for the same gene product, in particular for the same amino acidsequence, however, have a deviating sequence of bases due to thedegeneracy of the genetic code.

In a preferred embodiment, with regard to the adenoviruses according tothe present invention and the adenoviral replication system according tothe present invention and the use of them according to the presentinvention, respectively, the adenoviral nucleic acid is deficient forthe expression of the oncogene protein, particularly of the E1A protein,which means that it is either not coding for the 12S E1A protein or forthe 13S E1A protein, or it is neither coding for the 12S E1A protein northe 13S E1A protein, or is modified, as defined herein, and that theadenoviral replication system further comprises a nucleic acid of ahelper virus, whereby the nucleic acid of the helper virus comprises anucleic acid sequence which codes for the oncogene protein, inparticular for the E1A protein, which has the following characteristicsand imparts the following characteristics to the adenovirus,respectively, namely that it preferably is not replicating in YB-1nucleus-negative cells but in cells which are independent from the cellcycle YB-1 nucleus-positive, transactivating at least one viral gene, inparticular E1B55kDa, E4orf6, E4orf3 and/or E3ADP, in YB-1nucleus-positive cells, and/or does not translocate cellular YB-1 intothe nucleus. It is within the present invention that the transgenesdescribed herein are coded individually or together by the helper virusand/or expressed therefrom.

In an embodiment of such an adenoviral replication system according tothe present invention the adenoviral nucleic acid and/or the nucleicacid of the helper virus are furthermore present as vectors which arecapable of replicating.

It is within the present invention that the coding nucleic acid(s)coding for the adenoviruses which are used according to the presentinvention, is/are present in a vector, preferably in an expressionvector and this expression vector is used in accordance with the presentinvention.

In a further aspect the present invention is also related to a vectorgroup comprising at least two vectors, whereby the vector groupcomprises altogether an adenoviral replication system as describedherein, and the vector group is used in accordance with the presentinvention. It is intended that each of the components of the adenoviralreplication system is arranged on an individual vector, preferably anexpression vector.

Finally, the present invention is related in a further aspect to the useof a cell for the same purposes as described herein for theadenoviruses, whereby the cell comprises one or several nucleic acidswhich code for the adenoviruses described herein to be used inaccordance with the invention and/or a respective adenoviral replicationsystem and/or a respective vector and/or a vector group according to thepresent invention.

The previously described constructs of adenoviruses and in particulartheir nucleic acids and the nucleic acids coding therefor, may also beintroduced into a cell in parts, particularly into a tumor cell,whereupon due to the presence of the various individual components theymay act together such as if the individual components originated from asingle nucleic acid and a single or several adenoviruses, respectively.

The nucleic acids coding for adenoviruses, adenoviral systems or partsthereof, which are used in accordance with the invention, may be presentas vectors. Preferably, they are present as viral vectors. In case ofnucleic acids comprising adenoviral nucleic acids the virus particle ispreferably the vector. However, it is also within the invention thatsaid nucleic acids are present in a plasmid vector. In any case thevector comprises elements which provide for the propagation of theinserted nucleic acid, i. e. replication and optionally expression ofthe inserted nucleic acid, and control of them, respectively. Suitablevectors, in particular expression vectors, and corresponding elementsare known to the ones skilled in the art and, for example, described inGrunhaus, A., Horwitz, M. S., 1994, Adenoviruses as cloning vectors. InRice, C., edit., Seminars in Virology, London: Saunders ScientificPublications.

The aspect of the invention that is related to the vector group,accounts for the previously described embodiment, that the variouselements of the nucleic acid are not necessarily contained on one vectoronly. Accordingly, a vector group comprises at least two vectors.Otherwise, what has been said in relation to the vectors is alsoapplicable to the vectors and the vector group, respectively.

The adenoviruses which are used in accordance with the invention arecharacterised by various nucleic acids and gene products, respectively,disclosed herein, and may otherwise comprise all those elements known tothe one skilled in the art, as is also the case for adenoviruses of thewildtype (Shenk, T.: Adenoviridae: The virus and their replication.Fields Virology, 3^(rd) edition, edit. Fields, B. N., Knipe, D. M.,Howley, P. M. et al., Lippincott-Raven Publishers, Philadelphia, 1996,chapter 67).

The replication of adenoviruses is a very complex procedure and usuallymakes use of the human transcription factor E2F. During viral infection,first, the “early genes” E1, E2, E3 and E4 are expressed. The group ofthe “late genes” is responsible for the synthesis of the viralstructural proteins. For the activation of both the early as well as thelate genes, the E1 region consisting of the two transcriptional unitsE1A and E1B, which code for different E1A and E1B proteins, are criticalas the transcription of the E2, E3 and E4 is induced by them (Nevins, J.R., Cell 26, 213-220, 1981). Additionally, the E1A proteins can induceDNA synthesis in resting cells and thus initiate the entry into the Sphase (c. f. Boulanger and Blair, 1991). Additionally, they interactwith the tumor suppressors of the Rb class (Whyte, P. et al., Nature334, 124-127, 1988). In doing so, the cellular transcription factor E2Fis released. The E2F factors may subsequently bind to correspondingpromoter regions of both cellular as well as viral genes (in particularto the adenoviral E2 early promoter) and thus initiate transcription andreplication (Nevins, J. R., Science 258, 424-429, 1992).

The gene products of the E2 region are especially needed for theinitiation and performance, respectively, of the replication, as theycode for three essential proteins. The transcription of the E2 proteinsis controlled by two promoters, the “E2-early E2F-dependent” promoterwhich is also referred to herein as E2-early promoter or early E2promoter, and the “E2-late” promoter (Swaminathan and Thimmapaya, TheMolecular Repertoire of Adenoviruses III: Current Topics in Microbiologyand Immunology, Vol 199, 177-194, Springer Verlag 1995). Additionally,the products of the E4 region together with the E1A and E1B55kDa proteinplay an important role for the activity of E2F and the stability of p53,respectively. For example, the promoter is even more activated by adirect interaction of the E4orf6/7 protein coded by the E4 region, withthe heterodimer consisting of E2F and DP1 (Swaminathan and Thimmapaya,JBC 258, 736-746, 1996). Furthermore, p53 is inactivated by the complexconsisting of E1B55kDa and E4orf6 (Steegenga, W. T. et al., Oncogene 16,349-357, 1998), in order to successfully complete a lytic infectiouscycle. Additionally, the E1B55kDa protein exhibits a further importantfunction insofar as it promotes by interacting with the E4orf6 proteinthe export of viral RNA from the nucleus, whereas the proprietary RNAsof the cell are retained in the nucleus (Bridge and Ketner, Virology174, 345-353, 1990). A further important discovery is that the proteincomplex consisting of E1B55kDa/E4orf6 is localised in the so-called“viral inclusion bodies”. It is assumed that these structures are thesites of replication and transcription (Ornelles and Shenk, J. Virology65, 424-429, 1991).

A further region which is important for replication and in particularfor the release of adenoviruses, is the E3 region. The E3 regioncomprises more particularly the genetic information for a variety ofcomparatively small proteins which are not essential for the adenoviralinfectious cycle in vitro, i. e. are not essential in cell culture.However, they play an important role for the survival of the virusduring an acute and/or latent infection in vivo, as they have, amongothers, immune regulatory and apoptotic function(s) (Marshall S.Horwitz, Virololgie, 279, 1-8, 2001; Russell, supra). It could be shownthat a protein having a size of about 11.6 kDa induces cell death. Theprotein was, due to its function, referred to as ADP—for the englishterm adenovirus death protein—(Tollefson, J. Virology, 70, 2296-2306,1996). The protein is predominantly formed in the late phase of theinfectious cycle. Furthermore, overexpression of the protein results ina better lysis of the infected cells (Doronin et al., J. Virology, 74,6147-6155, 2000).

Furthermore, it is known to the present inventor that E1A deletedviruses, i. e. in particular viruses which do not have a 12S E1A proteinand which also do not express a 13S E1A protein, can very efficientlyreplicate at higher MOIs (Nevins J. R., Cell 26, 213-220, 1981), which,however, cannot be realised in any clinic application. This phenomenonis referred to as “E1A-like activity” in literature. It is was alsoknown that from the 5 proteins coded by E1A, two proteins, namely the12S and 13S protein, control and induce, respectively, expression of theother adenoviral genes (Nevins, J. R., Cell 26, 213-220, 1981;Boulanger, P. and Blair, E.; Biochem. J. 275, 281-299, 1991). Inconnection therewith it was shown that the transactivating function ispredominantly provided by the CR3 region of the 13S protein (Wong H Kund Ziff E B., J Virol., 68, 4910-20, 1994). Adenoviruses which havespecific deletions in the CR1 and/or CR2 region and/or CR3 region of the13S protein, are mostly replication-deficient, however, are stilltransactivating in some cell lines the viral genes and promoters,respectively, in particular the E2 region (Wong H K, Ziff E B., J Virol.68, 4910-20, 1994; Mymryk, J. S. and Bayley, S. T., Virus Research 33,89-97, 1994).

After infection of a cell, typically a tumor cell, using a wildtypeadenovirus, YB-1 is induced into the nucleus which is mediated by E1A,E1B55kDa and E4orf6, and is co-localised with E1B55kDa in the nucleus inthe viral inclusion bodies, which allows for an efficient replication ofthe virus in the cell nucleus both in vitro and in vivo. In connectiontherewith, it has already been found earlier that E4orf6 binds toE1B55kDa (Weigel, S. and Dobbelstein, M. J. Virology, 74, 764-772, 2000;Keith N. Leppard, Seminars in Virology, 8, 301-307, 1998) and thusmediates the transport and distribution, respectively, of E1B55kDa-intothe nucleus, which provides for an optimum virus production andadenoviral replication, respectively. An efficient replication of thevirus in accordance with the present invention is possible due to theinteraction of E1A, E1B55kDa and YB-1, and by the complex consisting ofE1B55kDa/E4orf6 with YB-1, respectively, and the co-localisation of YB-1and E1B55kDa in the nucleus in the so-called viral inclusion bodies andthus the use of the viruses described herein for replication in cellswhich are YB-1 nucleus-positive and for the manufacture of a medicamentfor the treatment of diseases, whereby YB-1 nucleus-positive cells areinvolved. The replication being thus possible with this cellularbackground, results in lysis of the cell, release of the virus andinfection and lysis of adjacent cells, so that in case of an infectionof a tumor cell and a tumor, respectively, finally lysis of the tumor,i. e. oncolysis, occurs.

YB-1 belongs to the group of highly conserved factors which bind to aninverted CAAT sequence, the so-called Y-box. They may be active in aregulatory manner both at the level of transcription as well astranslation (Wolfe, A. P. Trends in Cell Biology 8, 318-323, 1998). Agrowing number of Y-box dependant regulatory pathways is found in theactivation but also in the inhibition of growth and apoptosis associatedgenes (Swamynathan, S. K. et al., FASEB J. 12, 515-522, 1998).Accordingly, YB-1 directly interacts with p53 (Okamoto, T. et al.,Oncogene 19, 6194-6202, 2000), plays an important role in the geneexpression of Fas (Lasham, A. et al., Gene 252, 1-13, 2000), MDR and MRPgene expression (Stein, U. et al., JBC 276, 28562-69, 2001; Bargou, R.C. et al., Nature Medicine 3, 447-450, 1997) and in the activation oftopoisomerases and metalloproteinases (Mertens. P. R. et al., JBC 272,22905-22912, 1997; Shibao, K. et al., Int. J. Cancer 83, 732-737, 1999).Additionally, YB-1 is involved in the regulation of mRNA stability(Chen, C-Y. et al., Genes & Development 14, 1236-1248, 2000) and repairprocesses (Ohga, T. et al., Cancer Res. 56, 4224-4228, 1996).

The nuclear localisation of YB-1 in tumor cells results in E1Aindependent viral replication whereby in particular neither a 12S E1Aprotein nor a 13S E1A protein is present in an expressed form and used,respectively (Holm, P. S. et al. JBC 277, 10427-10434, 2002) and in caseof overexpression of the protein YB-1 in multidrug resistance (multipleresistance). Additionally it is known that the adenoviral proteins suchas e. g. E4orf6 and E1B55kDa have a positive effect on viral replication(Goodrum, F. D. and Ornelles, D. A, J. Virology 73, 7474-7488, 1999),whereby a functional E1A protein is responsible for switching on theother viral gene products (such as E4orf6, E3ADP and E1B55kDa) (NevinsJ. R., Cell 26, 213-220, 1981). This, however, does not occur with theE1A-minus adenoviruses of the prior art in which the 13S E1A protein isnot present. The nuclear localisation of YB-1 in multidrug resistantcells which have YB-1 in the nucleus, provides for replication andparticle formation, respectively, of such E1A-minus viruses. In thiscase, however, the efficiency of viral replication and particleformation is reduced by several multiples compared to wildtype Ad5. Acombination of YB-1 which is either already present in the nucleus ofthe tumor cell, or is induced into the tumor cell by external factors(e. g. application of cytostatics or irradiation or hyperthermia), i. e.is prompted to be present in the nucleus, particularly independent fromthe cell cycle, or is introduced as a transgene through a vector, with asystem, preferably with an adenoviral system, which switches onadenoviral genes, but which does not allow for viral replication, hasbeen surprisingly found to be a system which mediates a very efficientviral replication and particle formation through YB-1 and thus providesoncolysis. Suitable cytostatics are, among others, those which belong tothe following groups: anthracyclines, such as daunomycin and adriamycin;alkylating agents, such as cyclophosphamide; alkaloids, such asetoposide; vin-alkaloids, such as vincristine and vinblastine;antimetabolites such as for example 5-fluorouracil and methotrexate;platin derivatives, such as for example cis-platin; topoisomeraseinhibitors, such as camphothecine; and taxanes, such as for exampletaxole. The adenoviruses disclosed herein, in particular the recombinantadenoviruses, which are only capable of replicating in YB-1nucleus-positive cells, are limited in their capability to transactivatethe viral genes E1B55kDa E4orf6, E4orf3 and E3ADP, compared to thecorresponding transactivating capabilities of wildtype adenoviruses, inparticular wildtype Ad5. The present inventor has now surprisingly foundthat these limited transactivating capabilities may be compensated bythe corresponding genes and in particular by E1B55kDa and E4orf6 beingexpressed in combination with the nuclear localisation of YB-1. As shownin the examples herein, viral replication and particle formation,respectively, is increased under such conditions to a level which iscomparable to the replication and particle formation behaviour ofwildtype adenoviruses.

It is intended that the medicament in connection with which or for themanufacture of which the adenoviruses described herein are used inaccordance with the present invention, is usually appliedsystematically, although it is also within the present invention toapply or deliver such medicament locally. The application is done withthe intention that particularly those cells are infected with theadenovirus and that particularly in these cells replication occurs,which are involved, preferably in a causal manner, in the formation of acondition, typically a disease, for the diagnosis and/or preventionand/or treatment of which the medicament according to the presentinvention is used.

Such a medicament is preferably for the treatment of tumor diseases.Among the tumor diseases, those are particularly preferred in whicheither YB-1 is already located in the nucleus due to the mechanismunderlying the tumor disease, in particular the underlying pathologicalmechanism, or those where the presence of YB-1 in the nucleus is causedby exogenous measures, whereby the measures are suitable to transferYB-1 into the nucleus, induce YB-1 there or to express YB-1 there. Theterm tumor or tumor disease as used herein shall comprise both malignantas well as benign tumors and respective diseases. It can be intendedthat the medicament comprises at least one further pharmaceuticallyactive compound. The kind and the amount of such furtherpharmaceutically active compound will depend on the indication for whichthe medicament is to be used. In case the medicament is used for thetreatment and/or prevention of tumor diseases, typically cytostatics,such as for example cis-platin and taxol, daunoblastin, daunorubicin,adriamycin and/or mitoxantron or others of the cytostatics or groups ofcytostatics which are described herein, are used.

The medicament according to the present invention can be present invarious formulations, preferably in a liquid form. Furthermore, themedicament will contain stabilisers, buffers, preservatives and suchagents which are known to the one skilled in the art of pharmaceuticalformulations.

The present inventor has surprisingly found that the use in accordancewith the invention of the viruses described herein can be applied with avery high success rate to tumors which have YB-1 in the nucleusindependent from the cell cycle. Normally, YB-1 is located in thecytoplasm, in particular also in the perinuclear plasma. During S-phaseof the cell cycle, YB-1 can be found in the cell nucleus of both normalcells as well as tumor cells. This, however, is not sufficient toprovide for viral oncolysis using thus modified adenoviruses. Thecomparatively little efficacy of such attenuated adenoviruses describedin the prior art is ultimately based on their wrong application. Inother words, such adenoviral systems can be used, particularly also withan increased efficacy, where the molecular biological prerequisites forviral oncolysis are given when the attenuated or modified viruses asdescribed herein, are administered. In case of the describedadenoviruses which are to be used in accordance with the invention asdescribed herein, such as AdΔ24, d1922-947, E1Ad/01/07, CB016, d1520 andthe recombinant adenoviruses described in European patent EP 0 931 830,the prerequisites are given in case of tumor diseases the cells of whichshow a nuclear localisation of YB-1 independent of the cell cycle. Thisform of nuclear localisation may be either caused by the kind of tumoritself or may be caused by the measures or agents in accordance with theinvention which are described herein. The present invention thus definesa new group of tumors and tumor diseases, respectively, and thus also ofpatients, which can still be treated successfully with the viruses inaccordance with the invention, particularly also with the attenuated ormodified adenoviruses already described in the prior art.

A further group of patients which can be treated in accordance with thepresent invention using the adenoviruses, some of which are known andwhich can be used in accordance with the present invention, or using theadenoviruses which are described herein for the first time, inparticular using such adenoviruses which have mutations and deletions,respectively, in the E1A protein which do not interfere with the bindingof Rb/E2f, but which do not replicate in YB-1 nucleus-negative cells orwhich have and show, respectively, a strongly reduced replication asdefined herein, and/or a deleted oncoprotein, particularly E1A, such as,for example, the viruses AdΔ24, d1922-947, E1Ad/01/07, CB106 and theadenoviruses described in European patent EP 0 931 830, are thosepatients for which it is ensured that by applying or realising distinctconditions YB-1 is migrating into the nucleus or is induced there or istransported therein. The use of such adenoviruses in connection withthis group of patients is based on the finding that the induction ofviral replication is based on the nuclear localisation of YB-1 withsubsequent binding of YB-1 to the E2-late promoter. Due to the findingsdisclosed herein adenoviruses such as AdΔ24, d1922-947, E1Ad/01/07,CB106 and/or the adenoviruses described in European patent EP 0 931 830may also replicate in cells which are YB-1 nucleus-positive and/or incells in which YB-1 is deregulated as defined in the present invention.Insofar, these adenoviruses can, according to the present invention, beused for the treatment of diseases and groups of patients, respectively,which/who comprise cells having these characteristics, particularly whenthese cells are involved in the formation of the respective disease tobe treated. This is the basis for the success of AdΔ24, d1922-947,E1Ad/01/07, CB016 and the adenoviruses described in patent EP 0 931 830for the treatment of such tumors, in accordance with the presentinvention, which have YB-1 in the nucleus independent of the cell cycleor in which YB-1 is deregulated in the sense of the present disclosure.A further group of patients which can be treated in accordance with theinvention using the adenoviruses which are described herein as beingusable in accordance with the invention, and using those viruses, inparticular adenoviruses, which are described herein for the first time,are those which are YB-1 nucleus-positive and/or which are YB-1nucleus-positive as a result of the treatments described in thefollowing, whereby such treatment is preferably a medical treatment,and/or those patients which have undergone such treatment concomitantlywith the administration of respective viruses. It is within the presentinvention that YB-1 nucleus-positive patients are patients which haveYB-1 in the nucleus independent from the cell cycle in a number of cellsforming a tumor. Among these treatments is the administration ofcytostatics as described herein altogether and/or as used in a tumortherapy. Additionally, radiation, preferably radiation as used in atumor therapy, belongs to this group of treatments. Radiation means inparticular radiation with high energy radiation, preferably radioactiveradiation, preferably as used in tumor therapy. Hyperthermia and theapplication of hyperthermia, preferably hyperthermia as used in tumortherapy, are further treatments. In a particularly preferred embodimenthyperthermia is applied locally. Finally, hormone treatment,particularly hormone treatment as used in tumor therapy, is a furthertreatment. In connection with such hormone treatment anti-estrogensand/or anti-androgens are used Anti-estrogens such as tamoxifene,particularly in the therapy of breast cancer, and anti-androgens such asfor example flutamide or cyproterone acetate, are used in the therapy ofprostate cancer.

It is within the present invention that some of the cells forming thetumor comprise YB-1 either inherently or after induction and activeintroduction into the nucleus, respectively, or comprise deregulatedYB-1 in the sense of the present disclosure. Preferably, about 5% or anypercentage above, i. e. 6%, 7%, 8% etc. of the tumor forming cells aresuch YB-1 nucleus-positive cells or cells in which YB-1 is present in aderegulated manner. Nuclear localisation of YB-1 can be induced bystress applied from outside and by locally applied stress, respectively.This induction can, for example, occur by means of radiation, inparticular UV radiation, application of cytostatics, as, among others,already disclosed herein, and hyperthermia. In connection withhyperthermia it is essential that it can be realised now in a veryspecific manner, more particularly in a locally very specific manner,and may thus also provide for a specific nuclear localisation of YB-1 inthe cell nucleus and, because of this, provide the prerequisites for areplication of the adenovirus and thus for cell and tumor lysis, whichis preferably locally limited (Stein U, Jurchott K, Walther W, BergmannS, Schlag P M, Royer H D. J Biol Chem. 2001, 276(30):28562-9; Hu Z, JinS, Scotto K W. J Biol Chem. 2000 Jan. 28; 275(4):2979-85; Ohga T,Uchiumi T, Makino Y, Koike K, Wada M, Kuwano M, Kohno K. J Biol Chem.1998, 273(10:5997-6000).

The medicament according to the present invention could thus also beadministered to patients and groups of patients, or may be intended forthem, where through appropriate pretreatment or concomitant treatment atransport of YB-1 is affected, preferably in the respective tumor cells.

Based on this technical teaching it is for the person of the art withinhis skills to perform suitable modifications particularly on E1A which,for example, may comprise deletions or point mutations in order to thusgenerate various embodiments of the adenoviruses, which may be used inconnection with the use in accordance with the invention.

As has already been explained above, the adenoviruses which are used inaccordance with the present invention, are capable of replicating insuch cells and cellular systems, respectively, which have YB-1 in thenucleus. For answering the question whether the adenoviruses used inaccordance with the present invention are able to replicate and are thusable to lyse the tumor, the status of the cells with regard to thepresence or absence of Rb, i. e. the retinoblastome tumor suppressorproduct, is irrelevant. Additionally, it is in connection with the usein accordance with the invention of said adenoviruses, not essential totake into consideration the p53 status of the infected cells, the cellsto be infected or the cells to be treated, as by using the adenoviralsystems as disclosed herein in connection with YB-1 nucleus-positivecells, i. e. cells which have YB-1 in the nucleus irrespective of thecell cycle, this p53 status as well as the Rb status do not have animpact on the performance of the technical teaching disclosed herein.

The oncogene and oncogene protein, respectively, in particular E1A, canbe either under the control of the proprietor natural adenoviralpromoters and/or be controlled by means of a tumor or tissue specificpromoter. Suitable non-adenoviral promoters can be selected from thegroup comprising cytomegalovirus promoter, RSV (Rous sarcoma virus)promoter, adenovirus-based promoter Va I and the non-viral YB-1 promoter(Makino Y. et al., Nucleic Acids Res. 1996, 15, 1873-1878). Furtherpromoters which can be used in connection with each and any aspect ofthe invention disclosed herein, comprise the telomerase promoter, thealpha-fetoprotein (AFP) promoter, the caecinoembryonic antigen promoter(CEA) (Cao, G., Kuriyama, S., Gao, J., Mitoro, A., Cui, L., Nakatani,T., Zhang, X., Kikukawa, M., Pan, X., Fukui, H., Qi, Z. Int. J. Cancer,78, 242-247, 1998), the L-plastin promoter (Chung, I., Schwartz, P E.,Crystal, R C., Pizzorno, G, Leavitt, J., Deisseroth, A B. Cancer GeneTherapy, 6, 99-106, 1999), the arginine vasopressin promoter (Coulson, JM, Staley, J., Wall, P J. British J. Cancer, 80, 1935-1944, 1999), theE2f promoter (Tsukada et al. Cancer Res., 62, 3428-3477), the uroplakineII promoter (Zhang et al., Cancer Res., 62, 3743-3750, 2002) and the PSApromoter (Hallenbeck P L, Chang, Y N, Hay, C, Golightly, D., Stewart,D., Lin, J., Phipps, S., Chiang, Y L. Human Gene Therapy, 10, 1721-1733,1999). Furthermore, the YB-1 dependent E2-late promoter of adenovirusesas described in German patent application DE 101 50 984.7, is a promoterwhich can be used in the present invention.

It is known that the telomerase promoter is of crucial importance inhuman cells. Accordingly, telomerase activity is regulated throughtranscriptional control of the telomerase reverse transcriptase gene(hTERT), which is the catalytic subunit of the enzyme. The expression ofthe telomerase is active in 85% of human tumor cells. In contrastthereto, it is not active in most of the normal cells. Exempt therefromare germ cells and embryonic tissue (Braunstein, I. et al., CancerResearch, 61, 5529-5536, 2001; Majumdar, A. S. et al., Gene Therapy 8,568-578, 2001). More detailed studies on the hTERT promoter haverevealed that fragments of the promoters 283 bp and 82 bp, respectively,distant from the initiation codon are sufficient for specific expressionin tumor cells (Braunstein I. et al.; Majumdar A S at al., supra).Therefore, this promoter and the specific fragments, respectively, aresuitable to provide for a specific expression of a gene and particularlyof a transgene, preferably one of the transgenes disclosed herein, intumor cells only. The promoter shall allow the expression of themodified oncogene, preferably the E1A oncogene protein, in tumor cellsonly. Also, in a preferred embodiment, the expression of a transgene,particularly one which is selected from the group comprising E4orf5,E1B55kDa, ADP and YB-1, in such adenoviral vector is under the controlof any of these promoters. It is also within the present invention thatthe open reading frame of the transactivating oncogene protein, inparticular of the E1A protein, is in frame with one or several of thegene products of the adenoviral system. The open reading frame of thetransactivating E1A protein, however, can also be independent therefrom.

It is intended that with regard to the characteristics of the cells forthe lysis of which the adenoviruses described herein are used inaccordance with the present invention, these are, in an embodiment,resistant, preferably have a multidrug or multiple resistance.Resistance as used herein, refers preferably to a resistance against thecytostatics described herein. This multidrug resistance preferably goesalong with the expression, preferably an overexpression, of themembrane-bound transport protein P-glycoprotein which can be used as amarker for determining respective cells and can thus also be used fortumors and respective groups of patients having such multidrugresistance. The term resistance as used herein comprises both theP-glycoprotein mediated resistance which is also referred to asclassical resistance, as well as atypical resistance which comprisesresistance which is mediated through MRP, or other, non-P-glycoproteinmediated resistances. A further marker, which correlates with theexpression of YB-1, is topoisomerase II alpha. Insofar, in a screeningfor determining whether a patient may be treated with an expectation ofsuccess using the adenoviruses in accordance with the present invention,expression of topoisomerase II alpha can be used instead of or inaddition to the determination of YB-1 in the nucleus. A further markerwhich can basically be used in a manner similar as P-glycoprotein, isMRP. A further marker, at least to the extent that the colorectalcarcinoma cells or patients with colorectal carcinoma are concerned, isPCNA (engl. proliferating cell nuclear antigen) (Hasan S. et al.,Nature, 15, 387-391, 2001), as, for example, described by Shibao K. etal. (Shibao K et al., Int. Cancer, 83, 732-737, 1999). Finally, theexpression of MDR (multiple drug resistance) is a marker in theaforedescribed sense (Oda Y et al., Clin. Cancer Res., 4, 2273-2277,1998), at least for breast cancer cells and osteosarcoma cells. Afurther possible marker, which can be used in accordance with thepresent invention, is p73 (Kamiya, M., Nakazatp, Y., J Neurooncology 59,143-149 (2002); Stiewe et al., J. Biol. Chem., 278, 14230-14236, 2003).

It is thus a particular advantage of the present invention that alsopatients can be treated using the adenoviruses in accordance with thepresent invention, as described herein, which are otherwise deemed asbeing no longer treatable in the clinical sense and where a furthertreatment of the tumor disease according to the methods of the prior artis no longer possible with a reasonable expectation of success, inparticular where the use of cytostatics is no longer reasonably possibleand can no longer be successfully performed in the sense of influencingor reducing the tumor. The term tumor refers herein in general to eachand any tumor or cancer disease which either contains YB-1 in thenucleus inherently or contains YB-1 in the nucleus, preferablyindependent from the cell cycle, as a consequence of realising exogenousmeasures as described herein.

Additionally, the viruses described herein can be used for the treatmentof tumors in general. Preferably, these tumors are selected from thegroup comprising breast cancer, ovary carcinoma, prostate carcinoma,osteosarcoma, glioblastoma, melanoma, small cell lung carcinoma andcolorectal carcinoma. Further tumors are those which are resistant asdescribed herein, preferably those which are multiple resistant andparticularly also those tumors of the above described group.

The invention is related in a further aspect to a method for thescreening of patients which can be treated using one of the modifiedadenoviruses, i. e. an adenovirus as used in accordance with the presentinvention such as, for example, AdΔ24, d1922-947, E1Ad/01/07, CB016 orthe viruses described in European patent EP 0 931 830, whereby suchmethod comprises the following steps:

-   -   examining a sample of a tumor tissue and    -   determining whether YB-1 is located in the nucleus independent        from the cell cycle.

The presence of the afore-described markers can be detected instead ofor in addition to YB-1.

In case that the tumor tissue or a part thereof comprise YB-1 in thenucleus, in particular independent from cell cycle, the adenovirusesdisclosed therein, can be used in accordance with the practice of thepresent invention.

In an embodiment of the method according to the present invention theexamination of the tumor tissue is done by using an agent which isselected from the group comprising antibodies against YB-1, aptamersagainst YB-1 and spiegelmers against YB-1 as well as anticalines againstYB-1. Basically, the same means can be produced for the correspondingmarkers and used accordingly. The manufacture of antibodies, inparticular monoclonal antibodies, is known to the ones skilled in theart. A further means for specific detection of YB-1 or the markers, arepeptides which bind with a high affinity to the target structures, inthe present case YB-1 or said markers. In the prior art methods areknown such as phage-display in order to generate such peptides.Typically, a peptide library is taken as a starting point, wherebyindividual peptides have a length of from 8 to 20 amino acids and thesize of the library is about 10² to 10¹⁸, preferably 10⁸ to 10¹⁵different peptides. A special form of target molecule bindingpolypeptides are the so-called anticalines which are, for example,described in German patent application DE 197 42 706.

A further means for specific binding of YB-1 or the correspondingmarkers disclosed herein and thus for the detection of cell cyclusindependent localisation of YB-1 in the cellular nucleus, are theso-called aptamers, i.e. D-nucleic acids which are present either as RNAor DNA either as a single strand or a double strand and specificallybind to the target molecule. The generation of aptamers is, for example,described in European patent EP 0 533 838. A special form of aptamersare the so-called aptazymes, which, for example, are described byPiganeau, N. et al. (2000), Angew. Chem. Int. Ed., 39, no. 29, pages4369-4373. These are special embodiments of aptamers insofar as theycomprise apart from the aptamer part a ribozyme part and getcatalytically active upon binding or release of the target moleculebinding to the aptamer part and cleave a nucleic acid substrate whichgoes along with the generation of a signal.

A further form of aptamers are the so-called spiegelmers, i. e. targetmolecule binding nucleic acids which are made of L-nucleic acids. Themethod for the manufacture of such spiegelmers is, for example,described in WO 98/08856.

The sample of the tumor tissue can be obtained by puncture or throughsurgery. The assessment whether YB-1 is localised in the nucleusindependent from the cell cycle, is frequently done by using microscopictechniques and/or immuno histoanalysis, preferably using the antibody orany of the other aforementioned means. Further means for detecting YB-1in the nucleus and in particular for detecting that YB-1 is locatedthere independent from the cell cycle, are known to the one skilled inthe art. For example, the localisation of YB-1 can be easily detected instained tissue sections when screening them. The frequency of thepresence of YB-1 in the nucleus already indicates that the localisationis independent from the cell cycle. A further option for cell cycleindependent detection of YB-1 in the nucleus resides in the stainingagainst YB-1 and detection whether YB-1 is localised in the nucleus anddetermination of the phase of the cells. This as well as the detectionof YB-1 may also be performed by using the aforementioned means directedagainst YB-1. The detection of the means is done by methods known to theone skilled in the art. By said agents specifically binding to YB-1 andnot to any other structures within the sample to be analysed,particularly the cells, their localisation and because of their specificbinding to YB-1 also the localisation of YB-1 can be detected andestablished by a suitable labelling of the means. Methods for thelabelling of said means are known to the ones skilled in the art.

In the following, the present invention shall be further illustrated byreference to the figures and samples from which new features,embodiments and advantages may be taken.

FIG. 1 shows the structural design of the adenoviral vectors referred toas AdE1/E3-minus herein which are E1/E3-deleted adenoviruses, ofwildtype adenovirus and adenovirus d1520.

FIG. 2 shows the binding domains of the E1A protein with regard to thebinding of p300, p107 and p105.

FIG. 3 shows U2OS cells which do not have YB-1 in the nucleus, afterinfection with the E1/E3-deleted adenoviruses Ad5, referred to asE1/E3-minus Ad5, and d1520.

FIG. 4 shows 257RDB cells which have YB-1 in the nucleus, afterinfection with the E1/E3-deleted adenoviruses Ad5, referred to asE1/E3-minus Ad5, and adenovirus d1520.

FIG. 5 shows 257RDB cells and U2OS cells after infection with adenovirusd11119/1131.

FIG. 6 shows the result of an EMSA analysis which confirms that YB-1 ispresent in multidrug resistant cells and cell lines 257RDB, 181 RDB,MCF-7Ad, respectively, whereas YB-1 is not present in the nucleus ofU2OS and HeLa cells.

FIG. 7 shows the structural design of the E1A protein of wildtypeadenovirus, of adenovirus d1520 and adenovirus d11119/1131.

FIG. 8 is a column diagram showing the replication efficiency ofadenoviruses in the presence of additionally expressed viral proteins inabsolute figures.

FIG. 9 is a column diagram showing the increase of replicationefficiency of adenoviruses in the presence of additionally expressedviral proteins.

FIG. 10 shows wells grown with U2OS cells after crystal violet stainingand infection with d1520 with 10 and 30 pfu/cell, respectively, andcontrol (K) without administration of daunorubicine and with theadministration of 40 ng daunorubicin per ml, respectively.

FIG. 11 shows wells grown with HeLa cells, after crystal violet stainingand infection with d1520 and 10 and 30 pfu/cell and control (K),respectively, without administration of daunorubicine and administrationof 40 ng daunorubicine per ml, respectively.

FIG. 12 is a diagram of the tumor volume of tumors having differentorigins (RDB257 and HeLa) as a function of time after treatment with PBSand d1520, respectively.

FIG. 13 show pictures of sacrificed mice which developed a tumor basedon RDB257 cells after treatment with PBS and 5×10⁸ pfu d1520,respectively.

FIG. 14 is the result of a Southern Blot analysis of a cell extract (ofthe tumors grown subcutaneously) of RDB257 cells and HeLa cells afterinfection with d1520.

FIG. 15 is a column diagram showing the replication efficiency andparticle formation, respectively, of d1520 and wildtype adenoviruses inYB-1 nucleus-positive tumor cells (257RDB and 181RDB) and YB-1nucleus-negative tumor cells (HeLa, U2OS).

FIG. 16 shows the structural design of wildtype adenovirus andadenoviral vector AdXvir03.

FIG. 17 shows the structural design of adenoviral vector AdXvir03/01.

FIG. 18A/B shows wells grown with 181RDB cells (FIG. 18A) and 272RDBcells (FIG. 18B) after crystal violet staining and infection with Ad312(20 pfu/cell), Xvir03 (5 pfu/cell) and control (non-infected), wherebycrystal violet staining was performed five days past infection.

EXAMPLE 1 Types of E1A Modifications as May be Comprised by theAdenoviruses which are Used in Accordance with the Invention

FIG. 1 shows the structural design of adenoviral vectors AdE1/E3-minus,i. e. E1/E3-deleted adenoviruses, wildtype adenovirus and adenovirusd1520.

Adenovirus AdE1/E3-minus does not have a region coding for a functionalE1A or a functional E1B or E3 and is used in the present experiments asa control for toxicity.

Wildtype E1A gene codes for a total of 5 proteins which are generatedthrough alternative splicing of the E1A RNA. Among others, two differentproteins are generated, namely a 289 amino acid protein and a 243 aminoacid protein. d1520 does not code for the 289 amino acid protein as ithas a deletion in the CR3 stretch of the E1A gene which results in thelack of the 13S gene product. The adenovirus d1520 which may be used inaccordance with the invention is referred to as 12S-E1A virus by thoseskilled in the art. Adenovirus d1347 (Wong and Ziff, J. Virol., 68,4910-4920, 1994) known in the prior art is also a 12S-E1A virus whichcan be used in accordance with the present invention.

Within the 289 amino acid protein which is encoded by the 13S-E1A mRNA,there are 3 regions which are conserved among various adenoviralsubtypes. These are referred to as CR1, CR2 and CR3. While CR1 and CR2are present in both E1A proteins (E1A 12S and E1A 13S), i. e. in boththe 289 amino acid and the 243 amino acid protein, the CR3 region isonly present in the bigger one of the two aforementioned proteins.

The CR3 region is required for the activation of viral genes, inparticular of E1B, E2, E3 and E4. Viruses which only comprise thesmaller, i. e. 243 amino acid protein are only very weaklytransactivating the viral genes and do not promote adenoviralreplication in those cells which do not have YB-1 in the nucleus. AsYB-1 is present in the nucleus only in tumor cells and can be detectedonly there, this vector is suitable to induce tumor-specificreplication.

Due to the deletion of CR3 in d1520 this adenovirus cannot translocatecellular YB-1 into the cell's nucleus which is also referred to hereinas translocation, and is thus not in a position to replicate in cellswhich are YB-1 nucleus-negative and is thus a virus which can be used inaccordance with the present invention, whereby this virus comprises thetransactivation required in accordance with the present invention.

EXAMPLE 2 Mode of Action of Adenoviruses in Depending on the Rb Statusof Cells

FIG. 2 shows the binding domains of the E1A protein with regard to thebinding of p300, p107 and p105. P300, as well as p107, is a cellularbinding protein. The binding of the retinoblastoma protein (pRb), atumor suppressor protein, is mediated through CR1 and CR2. Studies haveshown that pRb and p107/p300 are in combination with the cellulartranscription factor E2F effective in regulating transcription. Thewildtype E1A protein interferes with the binding of E2F to Rb. The thusreleased E2F binds to the E2 early promoter and induces adenoviralreplication thereby.

It is known from the prior art that certain deletions in the E1Aoncoprotein may result in recombinant adenoviral vectors such as thosementioned in the following, which are capable of replicatingpredominantly in Rb-negative cells and can be used in accordance withthe present invention. For example, the adenoviral vector d1922-947comprises a deletion in the CR2 region (amino acid positions 122-129)and the vector CB016 has deletions in the CR1 region (amino acidpositions 27-80) and CR2 region (amino acid positions 122-129). Thevector E1Ad/01/07 comprises a deletion in the CR2 region (amino acidpositions 111-123). Additionally, because of an additional deletion atthe N-terminus (amino acid positions 4-25), additionally, there is nobinding to protein p300. The adenoviral vector AdΔ24 comprises adeletion in the CR2 region (amino acid positions 120-127). Theadenoviral vector described in patent EP 0 931 830 comprises deletionsin the CR1 region and CR2 region.

The binding mechanism of E2F/RB and the release of E2F mediated throughE1A is fundamentally different from the mechanism underlying the presentinvention. Unlike assumed in the prior art it is not the release of E2Ffrom the Rb protein which is essential, not to say critical for viralreplication, but it is the nuclear localisation of the humantranscription factor YB-1. This transcription factor is, in normalcells, only present in the cytoplasm over most of the cell cycle. Afterinfection with an adenovirus it is induced into the nucleus undercertain circumstances or is already present in the nucleus in distinctcellular systems, such as distinct tumor diseases including, forexample, but not limited thereto, breast cancer, ovary carcinoma,prostate carcinoma, osteosarcoma, glioblastoma, melanoma, small celllung carcinoma and colorectal carcinoma.

EXAMPLE 3 Infection of U2OS Cells

100,000 U2OS cells were plated per well. On the next day the cells wereinfected with the various adenoviruses as depicted in FIG. 3. Theinfection was performed in 500 μl serum free DMEM medium at 37° C. for 1h. Subsequently, the infection medium was removed and replaced by 2 mlcomplete medium (10% FCS/DMEM). The analysis was performed after 3 daysusing crystal violet staining.

As may be taken from FIG. 3, the U2OS cells which do not have YB-1 inthe nucleus, show no lysis as illustrated by crystal violet stainingafter infection with two different adenoviruses, namely theE1/E3-deleted adenovirus referred to as E1/E3-minus, and adenovirusd1520, which can be used in accordance with the present invention. Inconnection therewith, first, the medium is removed. Subsequently, thecells are overlaid with crystal violet (50% ETOH, 3% formaldehyde, 5%acetic acid, 1% crystal violet) and incubated at room temperature for5-10 min. Subsequently, the plates having 6 wells are thoroughly rinsedwith water and dried at room temperature.

This confirms the finding underlying the present invention that thepresence of YB-1 is required in order to induce the viruses used inaccordance with the present invention, to lyse the infected cells.

EXAMPLE 4 Infection of 257RDB Cells

100,000 257RDB cells were plated per well. On the next day the cellswere infected with the various adenoviruses as depicted in FIG. 4. Theinfection was performed in 500 μl serum free DMEM medium for 1 h at 37°C. Subsequently, the infection medium was removed and replaced by 2 mlcomplete medium (10% FCS/DMEM). The analysis was performed after threedays using crystal violet staining.

The result of this experiment is depicted in FIG. 4. The adenovirusreferred to as E1/E3-minus Ad5 which is E1/E3-deleted, did not show anylysis at low MOIs (pfu/cell) upon infection of 257RDB cells which haveYB-1 in the nucleus. In contrast thereto, d1520 which, as shown inexample 3, does not replicate in YB-1 nucleus-negative cells and at thesame time codes with E1A for a transactivating oncogene protein inaccordance with the present invention, results in a factually completelysis at an MOI (multiplicity of infection) of 40 pfu per cell and astill predominant lysis at an MOI of 10 pfu per cell. It can beconcluded therefrom that d1520 and similar viruses such as describedherein by d11119/1131 or AdXvir 03, require an MOI which is reduced byabout 1 magnitude (factor of ten) compared to E1-deleted or anE1/E3-deleted adenovirus which justifies their clinical use.

As depicted in FIG. 7, the protein E1A of d1520 is characterised in thatthe CR3 region thereof is deleted which results in the transactivationrequired for the use in accordance with the present invention andreplication in YB-1 nucleus-positive cells.

EXAMPLE 5 Infection of 257RDB and U2OS Cells with d11119/1131

As depicted in FIG. 5, there is no lysis at an MOI of 20 pfu per cellupon infection of YB-1 nucleus-negative U2OS cells with adenovirusd11119/1131 which exhibits a deletion of amino acids 4-138 of the E1Aprotein and the nucleic acid coding therefor, and further comprises astop codon after amino acid 218, whereby the expressed truncated E1Aprotein comprises the CR3 region of the complete E1A protein. As anegative control a non-infected cell layer was used.

In contrast thereto, there was factually a complete lysis of the celllayer at an MOI of 20 pfu per cell under the influence of adenovirusd11119/1131 in a cellular system such as 257RDB which contains YB-1 inthe nucleus, i. e. is YB-1 nucleus-positive. Insofar this example isanother proof that a modified E1A oncogene protein which, as depicted inFIG. 7, comprises, for example, only the CR3 region and which is lackingthe CR1 region and CR2 region, provides for the required transactivationin YB-1 nucleus-positive cells which is required for the replication ofadenoviruses in accordance with the present invention, which results inviral replication. The adenovirus d11119/1131 is thus a furtheradenovirus which can be used in accordance with the present invention.It is within the present invention that also viruses can be used whichare designed similar to d11119/1131 with regard to the CR3 region, but,in contrast thereto, have the CR1 region and/or CR2 region.

EXAMPLE 6 Detection of Nuclear YB-1 in Multidrug Resistant Cells

The example is based on the consideration that nuclear YB-1 should bindas a transcription factor to the Y-box (CAAT sequence) within the mdr1promoter (engl. multiple drug resistance promoter). In order to detectthis, a so-called EMSA analysis (electrophoretic mobility shift assay)was performed. In connection therewith, nuclear protein is isolated andsubsequently 1-10 μg protein is incubated together with a short DNAfragment (oligo) at 37° C. In order to determine nuclear YB-1, thefollowing oligonucleotide was used: mdr1 promoter in contrast to U2O3(Position −86 to −67): TGAGGCTGATTGGCTGGGCA (the X-box is underlined).

This DNA fragment is radioactively labelled at the 5′ end with ³²P priorto that. Subsequently, separation is performed in a native polyacrylamide-gel. In case the protein YB-1 is binding to a sequence in theoligonucleotide, this can be detected as any non-bound oligonucleotideis migrating faster in the gel than bound oligonucleotide (Holm, P. S.et al., JBC 277, 10427-10434, 2002; Bargou, R. C. et al., NatureMedicine 3, 447-450, 1997).

As depicted in FIG. 6, it could be shown with the EMSA analysis thatYB-1 is present in the nucleus of multidrug resistant cells 257RDB,181RDB and MCF-7Ad cells in contrast to cell lines U2OS and HeLa cells.

The results shown in example 4 and 5 confirm that the adenoviruses d1520and d11119/1131 replicate in YB-1 nucleus-positive cells such as, e. g.,257RDB in contrast to U205, and induce lysis thereof. This confirms thefinding about the use of the adenoviruses in accordance with the presentinvention. Additionally, the results confirm that already a, compared towildtype adenovirus, weak transactivation of viral genes in YB-1nucleus-positive cells through modified or deleted E1A gene productsresults in successful replication and lysis of such cells in thepresence of YB-1 in the nucleus, including, for example, multidrugresistant cells and that the adenoviruses as described herein, can thusbe used in the lysis of such tumors.

EXAMPLE 7 Increase of Replication Efficiency of E1-Minus Adenoviruses

This example shows that the early viral genes E1B55kDa and E4orf6 can besubstituted through transfection with the plasmid pE4orf6 and infectionwith the E1/E3-deleted adenovirus Ad-55K. Ad-55K is an E1/E3 deletedvirus, whereby E1B55kDa cloned into E1 and is under the control of CMV.This substitution is necessary with regard to the fact that AdYB-1, i.e. an adenovirus which expresses YB-1, does not express these earlygenes and that the present inventor has recognised that a substitutionof these early genes in a replication system which contains YB-1 in thenucleus, is capable of increasing replication efficiency and particleformation efficiency, respectively, to an extent comparable to the oneof wildtype adenoviruses of type Ad5.

The following was done:

Transfection of each 10⁵ U2OS cells with the plasmid pE4orf6 usinglipofectamine. The plasmid pE4orf6 carries the DNA sequence coding forthe early viral gene E4orf6 under the control of CMV.

24 h after transfection with the plasmid pE4orf6 the cells were infectedwith the YB-1 expressing E1/E3-deleted adenovirus AdYB-1 (50 pfu/cell)and the E1/E3-deleted E1B55kDa adenovirus Ad-55K (50 pfu/cell). Ad-55Kis an E1/E3-deleted virus which carries as transgene the viral geneEnder CMV control.

Subsequently, the cells were removed from the medium (2 ml) 5 days afterinfection (=post infectionem). The release of the viral particles fromthe isolated cells was done by alternating freezing and thawing forthree times (thaw/freeze). Subsequently, a plaque assay was performed on293 cells for determining the generated infectious particles (plaqueforming units per ml (pfu/ml)). The result is depicted in FIGS. 8 and 9.FIG. 8 shows the result of the plaque assay, represented in absolutefigures. The most significant difference compared to infection withAdYB-1 alone is shown by transfection with the plasmid pE4orf6 andco-infection with the two viruses AdYB-1 and Ad-55K. FIG. 9 shows theresult of FIG. 8, whereby the increase of the replication efficiency isrepresented as multifold of the replication determined for AdYB-1. Thecells infected with plasmid pE4orf6 and subsequently with AdYB-1 andE1B55kDa (Ad-55K) produced up to 25 times more pfu/ml.

Based on these results it can be concluded that the substitution ofE1B55kDa and E4orf6 increases the number of viruses formed (pfu/ml)after infection with the E1/E3-deleted adenovirus AdYB-1 by a factor ofup to 25. The additive effects of E1B55kDa and E4orf6 on the productionof plaque forming units (pfu) is significantly higher compared to theeffects of each of the two gene products.

Control experiments with one plasmid which expresses EGFP, clearlyshowed that in the experimental approach chosen only 10% of the cellswere successfully transfected with plasmid pE4orf6. The number of theparticles formed in the cells which express both E1B55kDa and E4orf6 iscomparable to the one of human adenovirus type 5 (wildtype). Thisconfirms the finding underlying the present invention that theexpression of E4orf6 and E1B55kDa is, in combination with the nuclearlocalisation of YB-1, able to provide for adenoviral replication andparticle formation, in particular of E1A-deleted adenoviruses, which iscomparable to the one of wildtype Ad5.

EXAMPLE 8 Increased Replication of Adenoviruses which are notReplicating in YB-1 Nucleus-Negative Cells, in YB-1 Nucleus-PositiveCells Upon Administration of Cytostatics

It is known in the prior art that the addition of different cytostaticsinduces nuclear localisation of the human transcription factor YB-1. Ashas been found by the present inventor, YB-1 localised in the nucleuscontrols adenoviral replication by means of activation of the adenoviralE2-late promoter. The combination of both effects can be used in orderto provide for specific tumor lysis.

In the practising of the oncolytic assays the following procedure wasfollowed: 200,000 cells (HeLa and U2OS, respectively) were plated intoeach well of a 6 well plate. On the next day 40 ng/ml (finalconcentration) of daunorubicine were added. After 3 hours of incubationthe cells were infected with 10 and 30 pfu d1520/cell, respectively.Subsequently, the cells were incubated in cytostatic free medium. After3-5 days the cells were stained using crystal violet.

As may be taken from FIGS. 10 and 11, the addition of daunorubicineinduces the replication of d1520 through nuclear localisation of YB-1.Thus, d1520 creates a bigger tumorlytic effect in combination with thecytostatic daunorubicine compared to daunorubicine alone.

EXAMPLE 9 In Vivo Tumor Lysis by d1520

The HeLa (YB-1 nucleus-negative) and 257RDB (YB-1 nucleus-positive)cells used in this in vivo study, were expanded under sterile cellculture conditions. Prior to the injection of the cells into mice(strain CD1NuNu) in order to generate a subcutaneous tumor, the cellsare harvested by trypsinisation, taken up in DMEM medium (10% FCS),counted and washed with PBS one time. Subsequently, the cells arecentrifuged, the PBS aspired and the cells are portioned in fresh PBSwith the desired cell number. The cell number which was subcutaneouslyinjected in this study, was each 5×10⁶ cells of both cell lines. Theinjection was performed subcutaneously into one flank of the animals,whereby HeLa cells were injected into the right side and 257RDB cellswere injected into the left side for better distinction. The growth ofthe tumors was controlled twice a week and thereby the length and thewidth of the tumors was measured using vernier calipers. Based thereon,the tumor volume was calculated based on the following mathematicalformula:¾π*a/2*(b/2)² a=length, b=width

Once the tumor has reached a volume of 200 to 520 mm³, the virus and PBSas negative control, respectively, were intratumorally applied. Thevolumes to be injected were identical and were 50 μl each time. This wasrepeated on 3 consecutive days. The overall dosage of applied viruseswas 5×10⁸ pfu. Subsequently, the tumor growth was continued to bedocumented twice a week and the volume was calculated. At the end of thestudy the mice were sacrificed and the tumors removed for furtheranalysis.

The results are depicted in FIGS. 12 and 13.

FIG. 12 shows a diagram representing the tumor volume as a function oftime and the various treatment schemes. In case the tumor was formed byRDB257, there was a significant growth of the tumor to about 438 mm³ to1466 mm³ upon injection of PBS. Under the influence of the vector d1520which was used in accordance with the invention, tumor growth could bereduced significantly. Starting from a mean tumor size of 344 mm³, thetumor size increased only by 21% to a total of 543 mm³.

In the present example the tumor consisting of HeLa cells was used as acontrol which upon administration of PBS behaved similarly to the RDB257based tumor upon administration of PBS. Tumors based on HeLa cells andtreated with d1520, however, still showed a significant increase intumor growth starting from 311 mm³ and increasing to 1954 mm³.

FIG. 13 shows a picture of the sacrificed nude mice which had a tumorgrown using RDB257. It can be clearly seen that after the application ofadenovirus d1520 in accordance with the present invention a significantreduction of the tumor occurred. In the present case there was even areduction in the tumor volume (day 1 after administration of virusd1520: 515 mm³; day 30 after administration of virus d1520: 350 mm³).

EXAMPLE 10 Southern Blot of Tumor DNA

DNA was extracted from a tumor sample which has been taken from themiddle of the tumor developed in example 9. For isolation the DneasyTissue Kit of Qiagen is used. The DNA isolation is done in accordancewith manufacturer's instructions. In accordance therewith, the DNA wasreleased from the cells through alkaline lysis. Subsequently, theisolated DNA is purified over a column. Subsequently, the concentrationof the isolated DNA is determined by photometry at 260 nm. The analysiswas performed using 2 μg of the DNA samples which were digested with 10units of restriction enzyme Kpn I. Subsequently, an electrophoreticseparation of the samples was performed in a 0.8% agarose gel.Subsequently, the DNA was blotted onto a nylon membrane (performedaccording to the system of Schleicher & Schuell). The DNA blotted ontothe membrane is hybridised against a specific 1501 bp DNA probe. The1501 bp DNA probe specifically binds to the 3369 bp Kpn I fragmentwithin the E2A coding Ad5 sequence. The probe was prepared by PCR(primer: 5′-GTC GGA GAT CAG ATC CGC GT, 5′-GAT CCT CGT CGT CTT CGC TT)and radioactively labelled using ³²P. Subsequently, the membrane iswashed and exposed to a film.

The result of the Southern Blot of tumor DNA is depicted in FIG. 14. Theanalysis confirms that only d1520 replicates in vitro in resistant cellsRDB257, as depicted in lanes 3, 4 and 5. Lane 1 shows as positivecontrol Ad-5d, lane 6, 7 and 8 show DNA from HeLa cells which wereinfected with d1520. As HeLa cells are not YB-1 nucleus positive thevirus d1520 did not replicate so that, in accordance therewith, the E2Asequence could not be detected.

A further result with d1520 is depicted in FIG. 15. Based on a plaqueassay the particle formation (pfu/ml) was investigated after infectionwith d1520 and wildtype adenovirus. Various YB-1 nucleus-positive(257RDB and 181RDB) tumor cells and YB-1 nucleus-negative tumor cellswere infected with d1520 and wildtype adenovirus.

The following procedure was practiced:

100,000-200,000 cells each were plated in so-called plates having 6wells (engl. 6 well plates) in L 15 medium (resistant cells) and DMEM(non-resistant cells) having 10% FCS. After 24 h infection with d1520and wildtype adenoviruses (10 pfu/cell) was performed. 3 days afterinfection (post infectionem) the viral particles were released from thecell suspension (3 ml) by alternating freezing and thawing for threetimes. Subsequently, a plaque assay was performed on 293 cells fordetermining the formed infectious particles (plaque forming units per ml(pfu/ml)). The result is depicted in FIG. 15. The result of the plaqueassay shows that d1520 is replicating in YB-1 nucleus-positive cells(257RDB and 181RDB) similar to wildtype adenovirus. Insofar areplication efficiency can be observed similar to the one of wildtypeadenoviruses when using, in accordance with the present invention, theadenoviruses described herein.

EXAMPLE 11 Structural Design of the Adenoviral Vector Xvir03

FIG. 16 shows the structural design of the adenoviral vector Xvir03. Theadenovirus Xvir03 is a so-called E1/E3-deleted adenovirus. This meansthat no E1A, E1B and E3 proteins are manufactured which are functionalin adenoviral replication. The deletion of the E1 region extends from342-3528; the deletion of the E3 region of the amino acid position27865-30995. As used herein, the term “E1-deleted virus” means a virusin which E1 is no longer functionally active. This can be achieved byinactivation with an otherwise mostly intact nucleic acid and amino acidsequence, however, can also mean a deletion of the E1 region codingproteins having various sizes. Because of the lack of the E1A and E1Bprotein and the nucleic acids coding therefor, the E4 region, such asE4orf6, is only weakly expressed (about 1-5% compared to wildtypeadenoviruses) or expressed not at all. The viral genes E1B55kDa andE4orf6 are expressed in the E1 region by means of the heterologous CMVpromoter (Clontech: Plasmid pShuttle) introduced into Xvir03. Instead ofthe CMV promoter each and any of the promoters as disclosed herein inconnection with the expression of E1A can be used. The open readingframe of both genes is linked with each other by means of a so-calledIRES sequence (engl. internal ribosomal entry site) (Pelletier, J. andSonenberg, N. Nature, 1988, 334, 320-325). This element (Novagen: pCITE)provides for the expression of 2 proteins from one mRNA

The Vector was Manufactured as Follows:

The plasmid E1B55kDa was created by cloning the open reading frame ofE1B55kDa from pCGNE1B from M. Dobelstein (University of Marburg) withXbaI and BfrI into the pShuttle vector from Clontech. Subsequently,E1B55kDa in pShuttle was linearised with ApaI, the ends blunt ended andcut with NheI.

In a second vector, pcDNA3.1 (+) (Invitrogen), subsequent to each otherthe IRES element as a PCR product was cloned with pCITE-4a(+) of thecompany Novagen as template by means of TA cloning into the EcoRVcleaving site, and the E4orf6 from the plasmid pCMV-E4orf6 (M.Dobelstein, University of Marburg) was cloned by means ofBamHI=IRES-E4orf6-pcDNA3.1(+). IRES-E4orf6 in pcDNA3.1(+) was linearisedwith NotI, the ends blunt ended and subsequently the fragmentIRES-E4orf6 was cut out with NheI. The fragment IRES-E4orf6 was linkedwith the open vector E1B55kDa-pShuttle (blunt, NheI). The cassette wassubsequently cloned from the E1B55kDa-IRES-E4orf6-pShuttle together withthe CMV promoter and the bovine growth hormone (BGH)-PolyA into the ΔE1,ΔE3 Adeno-X-Plasmid (Clontech) with I-Ceu I and PI-SceI, and referred toas AdcmvE1B/IRES/E4orf6. Subsequently, the adenovirus was made inaccordance with manufacturer's instructions (Clontech). The adenoplasmid which was linearised with PacI having the expression elementCMV-E155kDa-IRES-E4orf6-BGH polyA was transfected into HEK293 cells and11 days post transfectionem the ablating cells were removed togetherwith the medium in order to release the adenoviruses through repeatedfreeze-thaw cycles.

The vector described above is in principle suitable as are the otherviruses described herein for use in accordance with the presentinvention. In particular the afore-described vector is suitable toreplicate and trigger lysis insofar, in cells which are YB-1nucleus-positive cells as well as in cells where YB-1 is deregulated, i.e. is overexpressed compared to normal cells and non-tumor cells,respectively. The use of this vector particularly applies to thosediseases and groups of patients or collectives of patients which aredisclosed in connection with the other adenoviruses which are describedherein to be used in accordance with the present invention and the otheradenoviruses of the present invention disclosed herein.

EXAMPLE 12 Structural Design of the Adenoviral Vector Xvir03/01

As may be taken from FIG. 17, Xvir03/01 is a further development ofXvir03. Therapeutic genes such as, for example, the genes describedherein and the transgene can be cloned into the E3 region. Additionally,a deletion was introduced into the E4 region so as to avoid homologousrecombination with the E4orf6 from the expression cassette of Xvir03.This allows that larger transgenes can be cloned in this construct. Thedeleted E3 region contains SacI, NdeI and NheI restriction sites forintroducing a cassette, into which, for example, the therapeutictransgenes can be cloned.

Preparation of a Plasmid for Cloning Therapeutic Genes into the E3Region as Well as for Making Deletions in the E4 Region:

The pAdenoX-Plasmid of Clontech has a restriction site for SfuI behindthe 3′ ITR region which is absent in wildtype adenovirus. The E3-E4region was taken from pAdenoX (Clontech) with the SpeI (position 23644)and SfuI and transferred into pcDNA3.1(+)(Invitrogen)=pcDNA3.1-E3Δ27865-30995-E4. The majority of E4ORF6, namely33241-33875 was removed by means ofPstI=pcDNA3.1-E3Δ27865-30995,E4Δ33241-33875. For the further developmentof Xvir03 the deleted E3/E4 region frompcDNA3.1-E3Δ27865-30995,E4Δ33241-33875 was cloned by means of SfuI andSpeI into plasmid pAdenoX=pAdenoX E3Δ27865-30995,E4Δ33241-33875.

The expression cassette was subsequently, as described for Xvir03,cloned with I-Ceu I and PI-SceI from the E1B55kDa-IRES-E4orf6-pShuttletogether with the CMV promoter and the bovine growth hormone (BGH)-PolyAinto pAdenoX E3Δ27865-30995,E4Δ33241-33875 and referred to asAdcmvE1B/IRES/E4orf6-ΔE4 Subsequently, the adenovirus was made inaccordance with manufacturer's instructions (Clontech).

The afore-described vector is in principle useful as are the otherviruses described herein to be used in accordance with the presentinvention. In particular the afore-described vector is suitable toreplicate in YB-1 nucleus-positive cells as well as cells in which YB-1is deregulated, i. e. is overexpressed compared to normal cells andnon-tumor cells, and to cause lysis insofar. This vector can also beused for those diseases and groups of patients and collectives ofpatients which are disclosed herein for the other adenoviruses to beused in accordance with the present invention and the adenoviruses inaccordance with the present invention.

EXAMPLE 13 Oncolytic Effect of Xvir 03 in 257 RDB and 181 RDB Cells

100,000 cells (257RDB and 181RDB) were plated per well of a plate havingsix wells (engl.: 6 well plate). On the next day the cells were, asdepicted in FIG. 18, infected with Ad312 (20 pfu/cell) and Xvir03 (5pfu/cell). The infection was performed in 500 μl serum free DMEM mediumat 37° C. for 1 h. Subsequently, the infection medium was removed andreplaced by 2 ml complete medium (10% FCS/DMEM). The analysis was doneby means of crystal violet staining after 5 days. The result is depictedin FIGS. 18A and 18B.

As may be taken from FIGS. 18A and 18B, the multidrug resistant cellswhich have YB-1 in the nucleus, show lysis after infection with Ad312and Xvir03 only in case of Xvir03 as represented by the crystal violetstaining of the cells. In connection therewith, first the medium isremoved. Subsequently the cells are covered with crystal violet (50%ETOH, 3% formaldehyde, 5% acetic acid, 1% crystal violet) and incubatedat room temperature for 5-10 min. Subsequently, the six well plates arethoroughly rinsed with water and dried at room temperature.

It is known to the present inventor that E1A-deleted viruses (e. g.Ad312) which, however, are not transactivating adenoviruses in the senseof the present invention, may very efficiently replicate at higher MOIs(Nevins J. R., Cell 26, 213-220, 1981), which, however, cannot berealised in clinical application. This phenomenon is referred to in theliterature as “E1A-like activity”. The adenovirus Ad312 as used herein,is an E1A-deleted virus. At the titer used (20 pfu/cell), which is stillabove the clinically desirable titer, the early adenoviral genes such asE1B55kDa and E4orf6 are not expressed or expressed only to a very smallextent (Nevins J. R., Cell 26, 213-220, 1981). As already describedherein, these genes and proteins play an important role in viralreplication. In contrast thereto, these genes and proteins,respectively, are expressed by adenovirus Xvir03 (FIG. 16). As may betaken from FIGS. 18A and 18B, the expression of the genes E1B55kDa andE4orf6 will result in an efficient viral replication and cell lysis at aconcomitantly lower infection titer required (expressed as pfu/cell).This confirms the finding underlying the present invention, namely thatthe expression of E4orf6 and E1B55kDa and the absence of E1A) incombination with nuclear localisation of YB-1 is capable of inducing avery efficient adenoviral replication. The titer required therefor ofonly 1 to 5 pfu/cell now allows for clinical application.

This confirms the finding underlying the present invention, namely thatthe presence of YB-1 in the nucleus, particularly the presenceindependent from the cell cycle, is required in order to make theviruses which are to be used in accordance with the present invention,lyse infected cells.

The features of the invention disclosed in the preceding specification,the claims as well as the figures can both individually as well as inany combination be important to the realisation of the invention in itsvarious embodiments.

The invention claimed is:
 1. A pharmaceutical composition comprising anadenovirus, wherein the adenovirus expresses E1B55kDa and is replicationdeficient in cells that lack Y box binding protein 1 (“YB-1”) in thenucleus and replication competent in cells that have YB-1 in thenucleus; wherein the virus encodes an oncogene protein thattransactivates E1B55kDa, wherein the adenovirus is dl520 lacking afunctional CR3 domain; and wherein the pharmaceutical compositionfurther comprises a pharmaceutically active compound in addition todl520, wherein the additional pharmaceutically active compound isselected from the group consisting of a cytokine, a metalloproteinaseinhibitor, an angiogenesis inhibitor, a cytostatic, and a cell cycleinhibitor.
 2. The composition of claim 1, wherein the adenovirus iscapable of replicating in cells that are p53-positive or p53-negative.3. The composition of claim 1, wherein the oncogene protein is E1A. 4.The composition of claim 3, wherein the E1A does not induce nucleuslocalization of YB-1.
 5. The composition of claim 1, wherein theoncogene protein is encoded by a gene that is under the control of atissue and/or tumor specific promoter.
 6. The composition of claim 1,wherein the virus codes for YB-1.
 7. The composition of claim 6, whereinthe YB-1 is under the control of a tissue and/or tumor specificpromoter.
 8. The pharmaceutical composition of claim 1, wherein theoncogene protein additionally transactivates at least one adenoviralgene selected from the group consisting of E4orf6 and E3ADP.
 9. A methodfor the treatment of cancer, comprising administering to a subject inneed thereof the composition of claim
 1. 10. The method of claim 9,wherein the cancer is formed from a tumor, or part thereof, which isresistant to pharmacologically effective agents.
 11. The method of claim10, wherein the cells that form the tumor, or part thereof, overexpressmembrane-bound transport protein P glycoprotein.
 12. The method of claim9, wherein the oncogene protein is E1A.
 13. The method of claim 12,wherein the E1A does not induce nucleus localization of YB-1.
 14. Themethod of claim 9, wherein the oncogene protein is encoded by a genethat is under the control of a tissue and/ or tumor specific promoter.15. The composition of claim 1, wherein dl520 is E1B 19 K-minus.